U.S. patent application number 17/469549 was filed with the patent office on 2022-03-10 for pd-1 polypeptide variants.
This patent application is currently assigned to EUTILEX CO., LTD.. The applicant listed for this patent is EUTILEX CO., LTD.. Invention is credited to Jin Kyung CHOI, Seung Hee HAN, Sun Woo IM, Byoung S. KWON, Hanna LEE, Seunghyun LEE, Jin Sung PARK, Hyun Tae SON.
Application Number | 20220073586 17/469549 |
Document ID | / |
Family ID | 80469507 |
Filed Date | 2022-03-10 |
United States Patent
Application |
20220073586 |
Kind Code |
A1 |
KWON; Byoung S. ; et
al. |
March 10, 2022 |
PD-1 POLYPEPTIDE VARIANTS
Abstract
Provided are PD-1 polypeptide variants including an
extracellular domain that binds specifically to PD-L1, and a
transmembrane domain or a fragment thereof. The disclosure also
provides PD-1 Fc fusion proteins including an immunoglobulin Fc
region, and a PD-1 polypeptide variant.
Inventors: |
KWON; Byoung S.; (Seoul,
KR) ; LEE; Seunghyun; (Seoul, KR) ; LEE;
Hanna; (Seoul, KR) ; PARK; Jin Sung; (Seoul,
KR) ; CHOI; Jin Kyung; (Seoul, KR) ; HAN;
Seung Hee; (Seoul, KR) ; IM; Sun Woo; (Seoul,
KR) ; SON; Hyun Tae; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EUTILEX CO., LTD. |
Seoul |
|
KR |
|
|
Assignee: |
EUTILEX CO., LTD.
Seoul
KR
|
Family ID: |
80469507 |
Appl. No.: |
17/469549 |
Filed: |
September 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
63075641 |
Sep 8, 2020 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/30 20130101;
C07K 2317/31 20130101; A61P 35/00 20180101; C07K 2317/92 20130101;
C07K 2317/622 20130101; C07K 16/2878 20130101; C07K 2317/76
20130101; C07K 14/70503 20130101; C07K 2319/03 20130101; C07K
14/70521 20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; A61P 35/00 20060101 A61P035/00; C07K 16/28 20060101
C07K016/28 |
Claims
1. A programmed cell death 1 (PD-1) polypeptide variant comprising
an amino acid sequence having 95% or more sequence identity to SEQ
ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, wherein the PD-1
polypeptide variant comprises: an extracellular domain that binds
specifically to programmed cell death 1 ligand (PD-L1); and a
transmembrane domain or a fragment thereof.
2. The PD-1 polypeptide variant of claim 1, wherein the PD-1
polypeptide variant comprises an amino acid sequence of SEQ ID NO:
4, SEQ ID NO: 6, or SEQ ID NO: 8.
3. The PD-1 polypeptide variant of claim 1, wherein the
transmembrane domain comprises at least two amino acid
residues.
4. The PD-1 polypeptide variant of claim 1, wherein the polypeptide
variant has increased binding affinity to a PD-L1 molecule as
compared to a wild-type PD-1 polypeptide.
5. The PD-1 polypeptide variant of claim 4, wherein the polypeptide
variant has a binding affinity (K.sub.D) for a PD-L1 molecule of
about 1.times.10.sup.-8 to 1.times.10.sup.-10 M.
6. A programmed cell death 1 (PD-1) polypeptide variant comprising
an amino acid sequence having 95% or more sequence identity to
residues 24 to 172 of SEQ ID NO: 11, wherein said variant has a
mutation at least at one residue selected from the group consisting
of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93,
Q99, R114, L122, A125, A132, and R139.
7. The PD-1 polypeptide variant of claim 6, wherein said variant
comprises a mutation at D26, P34, V43, T45, T59, V64, L65, N66,
Y68, M70, N74, K78, Q99, L122, A125, A132, and R139.
8. The PD-1 polypeptide variant of claim 6, wherein said variant
comprises a mutation at D26, P34, V43, T45, T59, V64, L65, N66,
Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.
9. The PD-1 polypeptide variant of claim 6, wherein said mutations
are D26E, P34A, V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E,
N74G, K78T, C93H, Q99R, R114Q, L122Y, A125V, A132I, or R139G.
10. A PD-1 Fc fusion protein comprising: an immunoglobulin Fc
region; and a PD-1 polypeptide variant of claim 1 linked by a
peptide bond or a peptide linker sequence to the carboxy-terminus
of the immunoglobulin Fc region.
11. The PD-1 Fc fusion protein of claim 10, wherein the
immunoglobulin Fc region comprises an amino acid sequence having
95% or more sequence identity to SEQ ID NO: 10 or SEQ ID NO:
16.
12. The PD-1 Fc fusion protein of claim 10, wherein two copies of
PD-1 polypeptide variant is present, which may be the same or
different, and are linked by a peptide linker sequence.
13. The PD-1 Fc fusion protein of claim 10, wherein the fusion
protein has increased binding affinity to a PD-L1 molecule as
compared to a wild-type PD-1 polypeptide.
14. The PD-1 Fc fusion protein of claim 13, wherein the fusion
protein has a binding affinity (K.sub.D) for a PD-L1 molecule of
about 1.times.10.sup.-8 to 1.times.10.sup.-10 M.
15. A PD-1 Fc fusion protein comprising: an immunoglobulin Fc
region; and a PD-1 polypeptide variant of claim 6 linked by a
peptide bond or a peptide linker sequence to the carboxy-terminus
of the immunoglobulin Fc region.
16. The PD-1 Fc fusion protein of claim 15, wherein two copies of
PD-1 polypeptide variant is present, which may be the same or
different, and are linked by a peptide linker sequence.
17. A nucleic acid comprising: a sequence encoding a PD-1
polypeptide variant, wherein the polypeptide variant comprises a
sequence having 95% or more sequence identity to SEQ ID NO: 3, SEQ
ID NO: 5, or SEQ ID NO: 7.
18. The nucleic acid of claim 17, further comprising: a sequence
encoding an immunoglobulin Fc region, wherein the sequence encoding
the immunoglobulin Fc region comprises SEQ ID NO: 9.
19. An expression vector comprising the nucleic acid of claim
17.
20. The vector of claim 19, wherein the vector is a viral
vector.
21. A pharmaceutical composition comprising: the PD-1 polypeptide
variant of claim 1 or a fusion protein comprising an immunoglobulin
Fc region and said PD-1 polypeptide variant linked by a peptide
bond or a peptide linker sequence to the carboxy-terminus of the
immunoglobulin Fc region; and a pharmaceutically acceptable
carrier.
22. A method of treating a disease or a condition in a subject in
need thereof, the method comprising: administering to the subject
the pharmaceutical composition of claim 21, thereby treating a
disease or a condition.
23. The method of claim 22, wherein the subject has cancer.
24. The method of claim 23, wherein the cancer is selected from a
bladder cancer, breast cancer, cervical cancer, colon cancer,
endometrial cancer, esophageal cancer, fallopian tube cancer, gall
bladder cancer, gastrointestinal cancer, head and neck cancer,
hematological cancer, laryngeal cancer, liver cancer, lung cancer,
lymphoma, melanoma, mesothelioma, ovarian cancer, primary
peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer,
thyroid cancer, pancreatic cancer, renal cell carcinoma,
glioblastoma, and prostate cancer.
25. The method of claim 22, wherein when the pharmaceutical
composition comprises said fusion protein, decreased or no
antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC) effects are observed.
26. The method of claim 22, wherein when the pharmaceutical
composition comprises said fusion protein, decreased or no
hepatotoxicity is observed.
27. A pharmaceutical composition comprising: the PD-1 polypeptide
variant of claim 6 or a fusion protein comprising an immunoglobulin
Fc region and said PD-1 polypeptide variant linked by a peptide
bond or a peptide linker sequence to the carboxy-terminus of the
immunoglobulin Fc region; and a pharmaceutically acceptable
carrier.
28. A method of treating a disease or a condition in a subject in
need thereof, the method comprising: administering to the subject
the pharmaceutical composition of claim 27, thereby treating a
disease or a condition.
29. The method of claim 28, wherein the subject has cancer.
30. The method of claim 29, wherein the cancer is selected from a
bladder cancer, breast cancer, cervical cancer, colon cancer,
endometrial cancer, esophageal cancer, fallopian tube cancer, gall
bladder cancer, gastrointestinal cancer, head and neck cancer,
hematological cancer, laryngeal cancer, liver cancer, lung cancer,
lymphoma, melanoma, mesothelioma, ovarian cancer, primary
peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer,
thyroid cancer, pancreatic cancer, renal cell carcinoma,
glioblastoma, and prostate cancer.
31. The method of claim 28, wherein when the pharmaceutical
composition comprises said fusion protein, decreased or no
antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC) effects are observed.
32. The method of claim 28, wherein when the pharmaceutical
composition comprises said fusion protein, decreased or no
hepatotoxicity is observed.
33. A bispecific antibody comprising: an immunoglobulin Fc region;
a PD-1 polypeptide variant of claim 1 linked by a peptide bond or a
peptide linker sequence to the N-terminus of the immunoglobulin Fc
region; and a scFv for the anti-4-1BB antibody linked by a peptide
bond or a peptide linker sequence to the C-terminus of the
immunoglobulin Fc region, wherein the scFv for the anti-4-1BB
antibody comprises an amino acid sequence having 95% or more
sequence identity to an amino acid sequence comprising SEQ ID NO:
17 and 18 linked by a peptide bond or a peptide linker
sequence.
34. The bispecific antibody of claim 33, wherein the immunoglobulin
Fc region comprises an amino acid sequence having 95% or more
sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.
35. The bispecific antibody of claim 33, wherein the PD-1
polypeptide or the scFv or both are linked to the immunoglobulin Fc
region by a peptide linker selected from the group consisting of
SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
36. The bispecific antibody of claim 33, wherein the scFv for the
anti-4-1BB antibody comprises a sequence that is at least 90%
identical to SEQ ID NO: 22 or SEQ ID NO: 23.
37. The bispecific antibody of claim 33, wherein the scFv for the
anti-4-1BB antibody comprises SEQ ID NO: 22 or SEQ ID NO: 23.
38. The bispecific antibody of claim 33, wherein the bispecific
antibody has increased binding affinity to a PD-L1 molecule as
compared to a wild-type PD-1 polypeptide and specific binding
affinity to 4-1BB.
39. A pharmaceutical composition comprising: the bispecific
antibody of claim 33; and a pharmaceutically acceptable
carrier.
40. A method of treating a disease or a condition in a subject in
need thereof, the method comprising: administering to the subject
the pharmaceutical composition of claim 39, thereby treating a
disease or a condition.
41. The method of claim 40, wherein the subject has cancer.
42. The method of claim 41, wherein the cancer is selected from a
bladder cancer, breast cancer, cervical cancer, colon cancer,
endometrial cancer, esophageal cancer, fallopian tube cancer, gall
bladder cancer, gastrointestinal cancer, head and neck cancer,
hematological cancer, laryngeal cancer, liver cancer, lung cancer,
lymphoma, melanoma, mesothelioma, ovarian cancer, primary
peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer,
thyroid cancer, pancreatic cancer, renal cell carcinoma,
glioblastoma, and prostate cancer.
43. The method of claim 40, wherein when the pharmaceutical
composition comprises said fusion protein, decreased or no
antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC) effects are observed.
44. The method of claim 40, wherein when the pharmaceutical
composition comprises said fusion protein, decreased or no
hepatotoxicity is observed.
45. A bispecific antibody comprising: an immunoglobulin Fc region;
a PD-1 polypeptide variant of claim 6 linked by a peptide bond or a
peptide linker sequence to the N-terminus of the immunoglobulin Fc
region; and a scFv for the anti-4-1BB antibody linked by a peptide
bond or a peptide linker sequence to the C-terminus of the
immunoglobulin Fc region, wherein the scFv for the anti-4-1BB
antibody comprises an amino acid sequence having 95% or more
sequence identity to an amino acid sequence comprising SEQ ID NO:
17 and 18 linked by a peptide bond or a peptide linker
sequence.
46. The bispecific antibody of claim 45, wherein the immunoglobulin
Fc region comprises an amino acid sequence having 95% or more
sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.
47. The bispecific antibody of claim 45, wherein the PD-1
polypeptide or the scFv or both are linked to the immunoglobulin Fc
region by a peptide linker selected from the group consisting of
SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
48. The bispecific antibody of claim 45, wherein the scFv for the
anti-4-1BB antibody comprises a sequence that is at least 90%
identical to SEQ ID NO: 22 or SEQ ID NO: 23.
49. The bispecific antibody of claim 45, wherein the scFv for the
anti-4-1BB antibody comprises SEQ ID NO: 22 or SEQ ID NO: 23.
50. The bispecific antibody of claim 45, wherein the bispecific
antibody has increased binding affinity to a PD-L1 molecule as
compared to a wild-type PD-1 polypeptide and specific binding
affinity to 4-1BB.
51. A pharmaceutical composition comprising: the bispecific
antibody of claim 45; and a pharmaceutically acceptable
carrier.
52. A method of treating a disease or a condition in a subject in
need thereof, the method comprising: administering to the subject
the pharmaceutical composition of claim 51, thereby treating a
disease or a condition.
53. The method of claim 52, wherein the subject has cancer.
54. The method of claim 53, wherein the cancer is selected from a
bladder cancer, breast cancer, cervical cancer, colon cancer,
endometrial cancer, esophageal cancer, fallopian tube cancer, gall
bladder cancer, gastrointestinal cancer, head and neck cancer,
hematological cancer, laryngeal cancer, liver cancer, lung cancer,
lymphoma, melanoma, mesothelioma, ovarian cancer, primary
peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer,
thyroid cancer, pancreatic cancer, renal cell carcinoma,
glioblastoma, and prostate cancer.
55. The method of claim 54, wherein when the pharmaceutical
composition comprises said fusion protein, decreased or no
antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC) effects are observed.
56. The method of claim 54, wherein when the pharmaceutical
composition comprises said fusion protein, decreased or no
hepatotoxicity is observed.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. 63/075,641,
filed Sep. 8, 2020, the entire contents of which are incorporated
herein by reference.
BACKGROUND
[0002] Cancer remains one of the leading causes of death in the
world. Recent statistics report that 13% of the world population
dies from cancer. According to estimates from the International
Agency for Research on Cancer (IARC), in 2012 there were 14.1
million new cancer cases and 8.2 million cancer deaths worldwide.
By 2030, the global burden is expected to grow to 21.7 million new
cancer cases and 13 million cancer deaths due to population growth
and aging and exposure to risk factors such as smoking, unhealthy
diet and physical inactivity. Further, pain and medical expenses
for cancer treatment cause reduced quality of life for both cancer
patients and their families.
[0003] Programmed cell death protein 1 (PD-1) is an immune
checkpoint receptor with a role in regulating the immune system
response by down-regulating the immune system and promoting
self-tolerance by suppressing T cell inflammatory activity. Because
PD-1 is overexpressed in cancer, leading to increased T-cell
exhaustion and a diminished antitumor response, enhancing T cell
activation by blocking the PD-1/PD-L1 inhibitory pathway has great
potential for the treatment of diseases such as cancers.
SUMMARY
[0004] Provided herein are programmed cell death 1 (PD-1)
polypeptide variants comprising an amino acid sequence having 95%
or more sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID
NO: 8, wherein the PD-1 polypeptide variant comprises: an
extracellular domain that binds specifically to programmed cell
death 1 ligand (PD-L1); and a transmembrane domain or a fragment
thereof. In some embodiments, the PD-1 polypeptide variant
comprises an amino acid sequence having 97% or more sequence
identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In some
embodiments, the PD-1 polypeptide variant comprises an amino acid
sequence having 98% or more sequence identity to SEQ ID NO: 4, SEQ
ID NO: 6, or SEQ ID NO: 8. In some embodiments, the PD-1
polypeptide variant comprises an amino acid sequence of SEQ ID NO:
4, SEQ ID NO: 6, or SEQ ID NO: 8.
[0005] In some embodiments, the PD-1 polypeptide variant comprises
SEQ ID NO: 4. In some embodiments, the PD-1 polypeptide variant
comprises SEQ ID NO: 6. In some embodiments, the PD-1 polypeptide
variant comprises SEQ ID NO: 8.
[0006] The transmembrane domain as used in the present invention
may be the wild-type transmembrane domain of PD-1 corresponding to
amino acid residues 171-191 of SEQ ID NO: 11 or a fragment thereof.
In some embodiments, the transmembrane domain is a fragment of the
transmembrane domain including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid residues
inclusive of all ranges and sub-ranges bound by these values. For
example, the transmembrane domain fragment may comprises at least
two amino acid residues, at least 5 amino acid residues, or at
least 10 amino acid residues. In some embodiments, the
transmembrane domain has two amino acid residues corresponding to
amino acid residues 171-172 of SEQ ID NO: 11. In some embodiments,
the transmembrane domain has three amino acid residues
corresponding to amino acid residues 171-173 of SEQ ID NO: 11. In
some embodiments, the transmembrane domain has four amino acid
residues corresponding to amino acid residues 171-174 of SEQ ID NO:
11. In some embodiments, the transmembrane domain has five amino
acid residues corresponding to amino acid residues 171-175 of SEQ
ID NO: 11.
[0007] Included in the definition of the transmembrane domain or a
fragment thereof is a variant of the wild-type transmembrane domain
of PD-1 corresponding to amino acid residues 171-191 of SEQ ID NO:
11 modified by addition, deletion, substitution of one to five
amino acids inclusive of the integer values of one, two, three,
four, and five amino acids that may be modified, added, or
substituted relative to the wild-type transmembrane domain of PD-1
corresponding to amino acid residues 171-191 of SEQ ID NO: 11.
[0008] Provided herein are programmed cell death 1 (PD-1)
polypeptide variants comprising an amino acid sequence having 95%
or more sequence identity to residues 24 to 172 of SEQ ID NO: 11,
wherein said variant has a mutation at least at one residue
selected from the group consisting of D26, P34, V43, T45, T59, V64,
L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and
R139.
[0009] In some embodiments, the polypeptide variant has increased
binding affinity to a PD-L1 molecule as compared to a wild-type
PD-1 polypeptide. In some embodiments, the polypeptide variant has
a binding affinity (K.sub.D) for a PD-L1 molecule of about
1.times.10.sup.-8 to 1.times.10.sup.-10 M. In some embodiments, the
polypeptide variant has a binding affinity (K.sub.D) for a PD-L1
molecule of about 1.10.times.10.sup.-9M, about
1.037.times.10.sup.-9M, or about 7.14.times.10.sup.-10 M.
[0010] Also provided herein are PD-1 Fc fusion proteins comprising:
an immunoglobulin Fc region; and a PD-1 polypeptide variant linked
by a peptide bond or a peptide linker sequence to the
carboxy-terminus of the immunoglobulin Fc region, wherein the PD-1
polypeptide variant comprises an amino acid sequence having 95% or
more sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO:
8. In some embodiments, the immunoglobulin Fc region comprises SEQ
ID NO: 10 or SEQ ID NO: 16 or a sequence having 95% or more (e.g.,
96% or more, 97% or more, 98% or more, or 99% or more) sequence
identity to SEQ ID NO: 10 or SEQ ID NO: 16.
[0011] Also provided herein are PD-1 Fc fusion proteins comprising:
an immunoglobulin Fc region; and a PD-1 polypeptide variant linked
by a peptide bond or a peptide linker sequence to the
carboxy-terminus of the immunoglobulin Fc region, wherein the PD-1
polypeptide variant comprises an amino acid sequence having 95% or
more sequence identity to residues 24 to 172 of SEQ ID NO: 11,
wherein said variant has a mutation at least at one residue
selected from the group consisting of D26, P34, V43, T45, T59, V64,
L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and
R139.
[0012] Also provided herein are Fc fusion BsAb (Bispecific
antibody) comprising; an immunoglobulin Fc region; and a PD-1
polypeptide variant linked by a peptide bond or a peptide linker
sequence to the N-terminus of the immunoglobulin Fc region, wherein
the PD-1 polypeptide variant comprises an amino acid sequence
having 95% or more sequence identity to SEQ ID NO: 4, SEQ ID NO: 6,
or SEQ ID NO: 8; and a scFv for the anti-4-1BB antibody linked by a
peptide bond or a peptide linker sequence to the C-terminus of the
immunoglobulin Fc region, wherein the scFv for the anti-4-1BB
antibody comprises an amino acid sequence having 95% or more
sequence identity to an amino acid sequence comprising SEQ ID NO:
17 and 18 linked by a peptide bond or a peptide linker
sequence.
[0013] Also provided herein are Fc fusion BsAb (Bispecific
antibody) comprising; an immunoglobulin Fc region; and a PD-1
polypeptide variant linked by a peptide bond or a peptide linker
sequence to the N-terminus of the immunoglobulin Fc region, wherein
the PD-1 polypeptide variant comprises an amino acid sequence
having 95% or more sequence identity to residues 24 to 172 of SEQ
ID NO: 11, wherein said variant has a mutation at least at one
residue selected from the group consisting of D26, P34, V43, T45,
T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125,
A132, and R139; and a scFv for the anti-4-1BB antibody linked by a
peptide bond or a peptide linker sequence to the C-terminus of the
immunoglobulin Fc region, wherein the scFv for the anti-4-1BB
antibody comprises an amino acid sequence having 95% or more
sequence identity to an amino acid sequence comprising SEQ ID NO:
17 and 18 linked by a peptide bond or a peptide linker
sequence.
[0014] In some embodiments, the immunoglobulin Fc region comprises
SEQ ID NO: 10 or SEQ ID NO: 16 or a sequence having 95% or more
(e.g., 96% or more, 97% or more, 98% or more, or 99% or more)
sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16. In some
embodiments the scFv for the anti-4-1BB antibody is an amino acid
sequence that is at least 70% identical to SEQ ID NO: 22 or SEQ ID
NO: 23.
[0015] The BsAb antibody of the present invention has decreased or
no antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC) effects.
[0016] The BsAb antibody of the present invention demonstrates
decreased or no hepatotoxicity.
[0017] In some embodiments, the fusion protein has increased
binding affinity to a PD-L1 molecule as compared to a wild-type
PD-1 polypeptide. In some embodiments, the fusion protein has a
binding affinity (K.sub.D) for a PD-L1 molecule of about
1.times.10.sup.-8 to 1.times.10.sup.-10 M. In some embodiments, the
fusion protein has a binding affinity (K.sub.D) for a PD-L1
molecule of about 1.10.times.10.sup.-9M, about
1.037.times.10.sup.-9 M, or about 7.14.times.10.sup.-10 M.
[0018] Also provided herein are nucleic acids comprising: a
sequence encoding a PD-1 polypeptide variant, wherein the
polypeptide variant comprises a sequence having 95% or more
sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.
In some embodiments, the nucleic acid further comprises: a sequence
encoding an immunoglobulin Fc region, wherein the sequence encoding
the immunoglobulin Fc region comprises SEQ ID NO: 9.
[0019] Also provided herein are expression vectors comprising any
one of the nucleic acids provided herein. In some embodiments, the
vector is a viral vector.
[0020] Also provided herein are pharmaceutical compositions
comprising: any one of the PD-1 polypeptide variants provided
herein or any one of the PD-1 fusion proteins provided herein; and
a pharmaceutically acceptable carrier.
[0021] Also provided herein are methods of treating a subject, the
method comprising: administering to the subject in need thereof any
one of the pharmaceutical compositions provided herein, thereby
treating a disease or a condition. In some embodiments, the subject
has cancer. In some embodiments, the cancer is selected from a
bladder cancer, breast cancer, cervical cancer, colon cancer,
endometrial cancer, esophageal cancer, fallopian tube cancer, gall
bladder cancer, gastrointestinal cancer, head and neck cancer,
hematological cancer, laryngeal cancer, liver cancer, lung cancer,
lymphoma, melanoma, mesothelioma, ovarian cancer, primary
peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer,
thyroid cancer, pancreatic cancer, renal cell carcinoma,
glioblastoma, and prostate cancer.
BRIEF DESCRIPTION OF DRAWINGS
[0022] FIG. 1 shows the amino acid sequence of the human PD-1
protein (SEQ ID NO: 11).
[0023] FIG. 2 shows the sequence alignment for wild-type PD-1 (SEQ
ID NO: 2), PD-1.m7 (SEQ ID NO: 4), PD-1.m8 (SEQ ID NO: 6), and
euPD-1 (SEQ ID NO: 8).
[0024] FIG. 3 shows the structure of an exemplary recombinant
expression vector for PD-1 variant Fc fusion protein.
[0025] FIG. 4 shows SDS-PAGE results with PD-1 variant Fc fusion
proteins, PD-1 Fc fusion protein (1), PD-1.m7 Fc fusion protein
(2), PD-1.m8 Fc fusion protein (3), and euPD-1 Fc fusion protein
(4).
[0026] FIG. 5A-5E show a size exclusion chromatography graph with
gel filtration standard (FIG. 5A), wild-type PD-1 Fc fusion protein
(FIG. 5B), PD-1.m7 Fc fusion protein (FIG. 5C), PD-1.m8 Fc fusion
protein (FIG. 5D), and euPD-1 Fc fusion protein (FIG. 5E).
[0027] FIG. 6 shows the expression level of PD-L1 in PD-L1 high
expressed cell line MDA-MB-231 (human breast cancer cell) and PD-L1
low expressed cell line MCF-7 (human breast cancer cell) cells.
[0028] FIG. 7A shows FACS analysis of PD-1 Fc cell binding in PD-L1
positive cells.
[0029] FIG. 7B shows FACS analysis of PD-1 Fc cell binding in PD-L1
negative cells.
[0030] FIG. 8A and FIG. 8C show the assay methodology of Example
7.
[0031] FIG. 8B shows binding of euPD-1 Fc fusion protein, wild-type
PD-1 Fc fusion protein, and Tecentriq to antigen PD-L1.
[0032] FIG. 8D shows binding of euPD-1 Fc fusion protein, wild-type
PD-1 Fc fusion protein, and Tecentriq to antigen PD-L2.
[0033] FIG. 9A and FIG. 9B show the results of the results of the
PD-1/PD-L1 blockade bioassay in Example 8.
[0034] FIG. 10 shows the in vivo efficacy study control for Example
9.
[0035] FIG. 11A, FIG. 11B, FIG. 11C, and FIG. 11D show the result
of tumor size observation for euPD-1 Fc and tecentriq in Example
9.
[0036] FIG. 12 shows the liver toxicity index analysis in Example 9
(ALT: 17-77 U/L (FIG. 12A and FIG. 12E), AST: 54-298 U/L (FIG. 12B
and FIG. 12F), BUN: 8-33 mg/dL (FIG. 12C and FIG. 12G), T-BIL: 8-33
mg/dL (FIG. 12D and FIG. 12H)).
[0037] FIG. 13 shows Fc fusion BsAB using euPD-1 and anti-4-1BB
antibody described in Example 10.
[0038] FIG. 14 shows SDS-PAGE results with Fc fusion BsAB using
euPD-1 and anti-4-1BB antibody described in Example 10.
[0039] FIG. 15 shows a size exclusion chromatography graph with gel
filtration standard of the Fc fusion BsAB using euPD-1 and
anti-4-1BB antibody described in Example 10. Specifically, FIG. 15A
is a standard curve, FIG. 15B is euPD-1.times.94kvt HLC 218, FIG.
15C is euPD-1.times.94kvt LHC 218, FIG. 15D is euPD-1.2.times.94kvt
HLC 218, and FIG. 15E is euPD-1.2.times.94kvt LHC 218.
[0040] FIG. 16 shows the SPR result of the euPD-1 BsAB constructs
in Example 10.
[0041] FIG. 17A shows the antigen binding assay method used in
Example 11.
[0042] FIG. 17B shows the antigen binding result in Example 11.
[0043] FIG. 18 shows the results of the results of the 4-1BB/PD-1
combination bioassay in Example 12.
DETAILED DESCRIPTION
[0044] This disclosure describes variants of PD-1 polypeptides,
wherein the PD-1 polypeptide variants include mutations in the
wild-type PD-1 protein increasing affinity to PD-L1 molecules.
Definitions
[0045] About: The term "about", when used herein in reference to a
value, refers to a value that is similar, in context to the
referenced value. In general, those skilled in the art, familiar
with the context, will appreciate the relevant degree of variance
encompassed by "about" in that context. For example, in some
embodiments, the term "about" may encompass a range of values that
are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%,
10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred
value.
[0046] Administration: As used herein, the term "administration"
typically refers to the administration of a composition to a
subject or system to achieve delivery of an agent that is, or is
included in, the composition. Those of ordinary skill in the art
will be aware of a variety of routes that may, in appropriate
circumstances, be utilized for administration to a subject, for
example a human. For example, in some embodiments, administration
may be ocular, oral, parenteral, topical, etc. In some particular
embodiments, administration may be bronchial (e.g., by bronchial
instillation), buccal, dermal (which may be or comprise, for
example, one or more of topical to the dermis, intradermal,
interdermal, transdermal, etc.), enteral, intra-arterial,
intradermal, intragastric, intramedullary, intramuscular,
intranasal, intraperitoneal, intrathecal, intravenous,
intraventricular, within a specific organ (e. g. intrahepatic),
mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical,
tracheal (e.g., by intratracheal instillation), vaginal, vitreal,
etc. In some embodiments, administration may involve only a single
dose. In some embodiments, administration may involve application
of a fixed number of doses. In some embodiments, administration may
involve dosing that is intermittent (e.g., a plurality of doses
separated in time) and/or periodic (e.g., individual doses
separated by a common period of time) dosing. In some embodiments,
administration may involve continuous dosing (e.g., perfusion) for
at least a selected period of time.
[0047] Affinity: As is known in the art, "affinity" is a measure of
the strength a particular ligand binds to its partner. Affinities
can be measured in different ways. In some embodiments, affinity is
measured by a quantitative assay. In some such embodiments, binding
partner concentration may be fixed to be in excess of ligand
concentration so as to mimic physiological conditions.
Alternatively or additionally, in some embodiments, binding partner
concentration and/or ligand concentration may be varied. In some
such embodiments, affinity may be compared to a reference under
comparable conditions (e.g., concentrations).
[0048] Antibody agent: As used herein, the term "antibody agent"
refers to an agent that specifically binds to a particular antigen.
In some embodiments, the term encompasses any polypeptide or
polypeptide complex that includes immunoglobulin structural
elements sufficient to confer specific binding. Exemplary antibody
agents include, but are not limited to monoclonal antibodies,
polyclonal antibodies, and fragments thereof. In some embodiments,
an antibody agent may include one or more sequence elements are
humanized, primatized, chimeric, etc., as is known in the art. In
many embodiments, the term "antibody agent" is used to refer to one
or more of the art-known or developed constructs or formats for
utilizing antibody structural and functional features in
alternative presentation. For example, embodiments, an antibody
agent utilized in accordance with the present invention is in a
format selected from, but not limited to, intact IgA, IgG, IgE, or
IgM antibodies; bi- or multi-specific antibodies (e.g.,
Zybodies.RTM., etc.); antibody fragments such as Fab fragments,
Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and
isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc
fusions; single domain antibodies (e.g., shark single domain
antibodies such as IgNAR or fragments thereof); cameloid
antibodies; masked antibodies (e.g., Probodies.RTM.); Small Modular
ImmunoPharmaceuticals ("SMIPs.TM."); single chain or Tandem
diabodies (TandAb.RTM.); VHHs; Anticalins.RTM.; Nanobodies.RTM.
minibodies; BiTE.RTM.s; ankyrin repeat proteins or DARPINs.RTM.;
Avimers.RTM.; DARTs; TCR-like antibodies; Adnectins.RTM.;
Affilins.RTM.; Trans-Bodies.RTM.; Affibodies.RTM.; TrimerX.RTM.;
MicroProteins; Fynomers.RTM., Centyrins.RTM.; and KALBITOR.RTM.s.
In some embodiments, an antibody agent may lack a covalent
modification (e.g., attachment of a glycan) that it would have if
produced naturally. In some embodiments, an antibody agent may
contain a covalent modification (e.g., attachment of a glycan, a
payload [e.g., a detectable moiety, a therapeutic moiety, a
catalytic moiety, etc.], or other pendant group [e.g.,
poly-ethylene glycol, etc.]. In many embodiments, an antibody agent
is or comprises a polypeptide whose amino acid sequence includes
one or more structural elements recognized by those skilled in the
art as a complementarity determining region (CDR); in some
embodiments an antibody agent is or comprises a polypeptide whose
amino acid sequence includes at least one CDR (e.g., at least one
heavy chain CDR and/or at least one light chain CDR) that is
substantially identical to one found in a reference antibody. In
some embodiments an included CDR is substantially identical to a
reference CDR in that it is either identical in sequence or
contains between 1-5 amino acid substitutions as compared with the
reference CDR. In some embodiments an included CDR is substantially
identical to a reference CDR in that it shows at least 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity with the reference CDR. In some embodiments
an included CDR is substantially identical to a reference CDR in
that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence
identity with the reference CDR. In some embodiments an included
CDR is substantially identical to a reference CDR in that at least
one amino acid within the included CDR is deleted, added, or
substituted as compared with the reference CDR but the included CDR
has an amino acid sequence that is otherwise identical with that of
the reference CDR. In some embodiments an included CDR is
substantially identical to a reference CDR in that 1-5 amino acids
within the included CDR are deleted, added, or substituted as
compared with the reference CDR but the included CDR has an amino
acid sequence that is otherwise identical to the reference CDR. In
some embodiments an included CDR is substantially identical to a
reference CDR in that at least one amino acid within the included
CDR is substituted as compared with the reference CDR but the
included CDR has an amino acid sequence that is otherwise identical
with that of the reference CDR. In some embodiments an included CDR
is substantially identical to a reference CDR in that 1-5 amino
acids within the included CDR are deleted, added, or substituted as
compared with the reference CDR but the included CDR has an amino
acid sequence that is otherwise identical to the reference CDR. In
some embodiments, an antibody agent is or comprises a polypeptide
whose amino acid sequence includes structural elements recognized
by those skilled in the art as an immunoglobulin variable domain.
In some embodiments, an antibody agent is a polypeptide protein
having a binding domain which is homologous or largely homologous
to an immunoglobulin-binding domain. In some embodiments, an
antibody agent is or comprises at least a portion of a chimeric
antigen receptor (CAR).
[0049] Antigen: The term "antigen", as used herein, refers to an
agent that binds to an antibody agent. In some embodiments, an
antigen binds to an antibody agent and may or may not induce a
particular physiological response in an organism. In general, an
antigen may be or include any chemical entity such as, for example,
a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a
lipid, a polymer (including biologic polymers [e.g., nucleic acid
and/or amino acid polymers] and polymers other than biologic
polymers [e.g., other than a nucleic acid or amino acid polymer])
etc. In some embodiments, an antigen is or comprises a polypeptide.
In some embodiments, an antigen is or comprises a glycan. Those of
ordinary skill in the art will appreciate that, in general, an
antigen may be provided in isolated or pure form, or alternatively
may be provided in crude form (e.g., together with other materials,
for example in an extract such as a cellular extract or other
relatively crude preparation of an antigen-containing source). In
some certain embodiments, an antigen is present in a cellular
context (e.g., an antigen is expressed on the surface of a cell or
expressed in a cell). In some embodiments, an antigen is a
recombinant antigen.
[0050] Antigen binding domain: As used herein, refers to an
antibody agent or portion thereof that specifically binds to a
target moiety or entity. Typically, the interaction between an
antigen binding domain and its target is non-covalent. In some
embodiments, a target moiety or entity can be of any chemical class
including, for example, a carbohydrate, a lipid, a nucleic acid, a
metal, a polypeptide, or a small molecule. In some embodiments, an
antigen binding domain may be or comprise a polypeptide (or complex
thereof). In some embodiments, an antigen binding domain is part of
a fusion polypeptide. In some embodiments, an antigen binding
domain is part of a chimeric antigen receptor (CAR).
[0051] Associated with: Two events or entities are "associated"
with one another, as that term is used herein, if the presence,
level, and/or form of one is correlated with that of the other. For
example, a particular entity (e.g., polypeptide, genetic signature,
metabolite, microbe, etc.) is considered to be associated with a
particular disease, disorder, or condition, if its presence, level
and/or form correlates with incidence of and/or susceptibility to
the disease, disorder, or condition (e.g., across a relevant
population). In some embodiments, two or more entities are
physically "associated" with one another if they interact, directly
or indirectly, so that they are and/or remain in physical proximity
with one another. In some embodiments, two or more entities that
are physically associated with one another are covalently linked to
one another; in some embodiments, two or more entities that are
physically associated with one another are not covalently linked to
one another but are non-covalently associated, for example by means
of hydrogen bonds, van der Waals interaction, hydrophobic
interactions, magnetism, and combinations thereof.
[0052] Binding: It will be understood that the term "binding", as
used herein, typically refers to a non-covalent association between
or among two or more entities. "Direct" binding involves physical
contact between entities or moieties; indirect binding involves
physical interaction by way of physical contact with one or more
intermediate entities. Binding between two or more entities can
typically be assessed in any of a variety of contexts--including
where interacting entities or moieties are studied in isolation or
in the context of more complex systems (e.g., while covalently or
otherwise associated with a carrier entity and/or in a biological
system or cell).
[0053] Cancer: The terms "cancer", "malignancy", "neoplasm",
"tumor", and "carcinoma", are used herein to refer to cells that
exhibit relatively abnormal, uncontrolled, and/or autonomous
growth, so that they exhibit an aberrant growth phenotype
characterized by a significant loss of control of cell
proliferation. In some embodiments, a tumor may be or comprise
cells that are precancerous (e.g., benign), malignant,
pre-metastatic, metastatic, and/or non-metastatic. The present
disclosure specifically identifies certain cancers to which its
teachings may be particularly relevant. In some embodiments, a
relevant cancer may be characterized by a solid tumor. In some
embodiments, a relevant cancer may be characterized by a
hematologic tumor. In general, examples of different types of
cancers known in the art include, for example, hematopoietic
cancers including leukemias, lymphomas (Hodgkin's and
non-Hodgkin's), myelomas and myeloproliferative disorders;
sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous
cell carcinomas of the mouth, throat, larynx, and lung, liver
cancer, genitourinary cancers such as prostate, cervical, bladder,
uterine, and endometrial cancer and renal cell carcinomas, bone
cancer, pancreatic cancer, skin cancer, cutaneous or intraocular
melanoma, cancer of the endocrine system, cancer of the thyroid
gland, cancer of the parathyroid gland, head and neck cancers,
breast cancer, gastro-intestinal cancers and nervous system
cancers, benign lesions such as papillomas, and the like.
[0054] Chemotherapeutic Agent: The term "chemotherapeutic agent",
has used herein has its art-understood meaning referring to one or
more pro-apoptotic, cytostatic and/or cytotoxic agents, for example
specifically including agents utilized and/or recommended for use
in treating one or more diseases, disorders or conditions
associated with undesirable cell proliferation. In many
embodiments, chemotherapeutic agents are useful in the treatment of
cancer. In some embodiments, a chemotherapeutic agent may be or
comprise one or more alkylating agents, one or more anthracyclines,
one or more cytoskeletal disruptors (e.g. microtubule targeting
agents such as taxanes, maytansine and analogs thereof, of), one or
more epothilones, one or more histone deacetylase inhibitors
HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of
topoisomerase I and/or topoisomerase II), one or more kinase
inhibitors, one or more nucleotide analogs or nucleotide precursor
analogs, one or more peptide antibiotics, one or more
platinum-based agents, one or more retinoids, one or more vinca
alkaloids, and/or one or more analogs of one or more of the
following (i.e., that share a relevant anti-proliferative
activity). In some particular embodiments, a chemotherapeutic agent
may be or comprise one or more of Actinomycin, All-trans retinoic
acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin,
Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil,
Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel,
Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide,
Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib,
Irinotecan, Maytansine and/or analogs thereof (e.g. DM1)
Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a
Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide,
Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine,
Vindesine, Vinorelbine, and combinations thereof. In some
embodiments, a chemotherapeutic agent may be utilized in the
context of an antibody-drug conjugate. In some embodiments, a
chemotherapeutic agent is one found in an antibody-drug conjugate
selected from the group consisting of: hLL1-doxorubicin,
hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38,
hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox,
hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox,
P4/D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin,
trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab
vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A,
AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F,
MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593,
RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, vorsetuzumab
mafodotin, and lorvotuzumab mertansine.
[0055] Engineered: In general, the term "engineered" refers to the
aspect of having been manipulated by the hand of man. For example,
a polypeptide is considered to be "engineered" when the polypeptide
sequence manipulated by the hand of man. For example, in some
embodiments of the present invention, an engineered polypeptide
comprises a sequence that includes one or more amino acid
mutations, deletions and/or insertions that have been introduced by
the hand of man into a reference polypeptide sequence. In some
embodiments, an engineered polypeptide includes a polypeptide that
has been fused (i.e., covalently linked) to one or more additional
polypeptides by the hand of man, to form a fusion polypeptide that
would not naturally occur in vivo. Comparably, a cell or organism
is considered to be "engineered" if it has been manipulated so that
its genetic information is altered (e.g., new genetic material not
previously present has been introduced, for example by
transformation, mating, somatic hybridization, transfection,
transduction, or other mechanism, or previously present genetic
material is altered or removed, for example by substitution or
deletion mutation, or by mating protocols). As is common practice
and is understood by those in the art, derivatives and/or progeny
of an engineered polypeptide or cell are typically still referred
to as "engineered" even though the actual manipulation was
performed on a prior entity.
[0056] Pharmaceutical composition: As used herein, the term
"pharmaceutical composition" refers to a composition in which an
active agent is formulated together with one or more
pharmaceutically acceptable carriers. In some embodiments, the
composition is suitable for administration to a human or animal
subject. In some embodiments, the active agent is present in unit
dose amount appropriate for administration in a therapeutic regimen
that shows a statistically significant probability of achieving a
predetermined therapeutic effect when administered to a relevant
population.
[0057] Pharmaceutically acceptable carrier: The term
"pharmaceutically acceptable carrier", as used herein, generally
has its art-recognized meaning of a pharmaceutically acceptable
material, composition or carrier, such as a liquid or solid filler,
stabilizer, dispersing agent, suspending agent, diluent, excipient,
thickening agent, solvent or encapsulating material, involved in
carrying or transporting a compound useful within the invention
within or to the patient such that it may perform its intended
function. Typically, such constructs are carried or transported
from one organ, or portion of the body, to another organ, or
portion of the body. Each carrier must be "acceptable" in the sense
of being compatible with the other ingredients of the formulation,
including the compound useful within the invention, and not
injurious to the patient. As used herein, "pharmaceutically
acceptable carrier" also includes any and all coatings,
antibacterial and antifungal agents, and absorption delaying
agents, and the like that are compatible with the activity of the
compound useful within the invention, and are physiologically
acceptable to the patient. The term "pharmaceutically acceptable
carrier" may further include a pharmaceutically acceptable salt of
the compound useful within the invention. Other additional
ingredients that may be included in the pharmaceutical compositions
used in the practice of the invention are described, for example,
in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack
Publishing Co., 1985, Easton, Pa.), which is incorporated herein by
reference. The "pharmaceutically acceptable carrier" is useful for
the preparation of a pharmaceutical composition that is: generally
compatible with the other ingredients of the composition, not
deleterious to the recipient, and neither biologically nor
otherwise undesirable. "A pharmaceutically acceptable carrier"
includes one or more than one carrier. Embodiments include carriers
for topical, ocular, parenteral, intravenous, intraperitoneal
intramuscular, sublingual, nasal or oral administration.
"Pharmaceutically acceptable carrier" also includes agents for
preparation of aqueous dispersions and sterile powders for
injection or dispersions.
[0058] Pharmaceutically acceptable salt: As used herein, the term
"pharmaceutically acceptable salt" generally has its art-recognized
meaning and refers to derivatives of the compounds provided herein
wherein the parent compound is modified by converting an existing
acid or base moiety to its salt form. Examples of pharmaceutically
acceptable salts include, but are not limited to, mineral or
organic acid salts of basic residues such as amines; alkali or
organic salts of acidic residues such as carboxylic acids; and the
like. The pharmaceutically acceptable salts of the compounds
provided herein include the conventional non-toxic salts of the
parent compound formed, for example, from non-toxic inorganic or
organic acids. The pharmaceutically acceptable salts of the
compounds provided herein can be synthesized from the parent
compound which contains a basic or acidic moiety by conventional
chemical methods. Generally, such salts can be prepared by
combining the free acid or base forms of these compounds with a
stoichiometric amount of the appropriate base or acid in water or
in an organic solvent, or in a mixture of the two; generally,
nonaqueous media such as ether, ethyl acetate, ethanol,
isopropanol, or acetonitrile may be used. Lists of suitable salts
are found in Remington's Pharmaceutical Sciences, 17.sup.th ed.,
Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of
Pharmaceutical Science, 66, 2 (1977), each of which is incorporated
herein by reference in its entirety.
[0059] Excipient: As used herein, the term "excipient" generally
has its art-recognized meaning and refers to physiologically
compatible additives useful in preparation of a pharmaceutical
composition. Examples of pharmaceutically acceptable carriers and
excipients can, for example, be found in Remington Pharmaceutical
Science, 16.sup.th Ed.
[0060] Polypeptide: The term "polypeptide", as used herein,
generally has its art-recognized meaning of a polymer of at least
three amino acids. Those of ordinary skill in the art will
appreciate that the term "polypeptide" is intended to be
sufficiently general as to encompass not only polypeptides having a
complete sequence recited herein, but also to encompass
polypeptides that represent functional fragments (i.e., fragments
retaining at least one activity) of such complete polypeptides.
Moreover, those of ordinary skill in the art understand that
protein sequences generally tolerate some substitution without
destroying activity. Thus, any polypeptide that retains activity
and shares at least about 30-40% overall sequence identity, often
greater than about 50%, 60%, 70%, or 80%, and further usually
including at least one region of much higher identity, often
greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more
highly conserved regions, usually encompassing at least 3-4 and
often up to 20 or more amino acids, with another polypeptide of the
same class, is encompassed within the relevant term "polypeptide"
as used herein. Polypeptides may contain L-amino acids, D-amino
acids, or both and may contain any of a variety of amino acid
modifications or analogs known in the art. Useful modifications
include, e.g., terminal acetylation, amidation, methylation, etc.
In some embodiments, proteins may comprise natural amino acids,
non-natural amino acids, synthetic amino acids, and combinations
thereof. The term "peptide" is generally used to refer to a
polypeptide having a length of less than about 100 amino acids,
less than about 50 amino acids, less than 20 amino acids, or less
than 10 amino acids. In some embodiments, proteins are antibody
agents, antibody fragments, biologically active portions thereof,
and/or characteristic portions thereof.
[0061] Recombinant: as used herein, is intended to refer to
polypeptides that are designed, engineered, prepared, expressed,
created, manufactured, and/or or isolated by recombinant means,
such as polypeptides expressed using a recombinant expression
vector transfected into a host cell; polypeptides isolated from a
recombinant, combinatorial human polypeptide library; polypeptides
isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.)
that is transgenic for or otherwise has been manipulated to express
a gene or genes, or gene components that encode and/or direct
expression of the polypeptide or one or more component(s),
portion(s), element(s), or domain(s) thereof; and/or polypeptides
prepared, expressed, created or isolated by any other means that
involves splicing or ligating selected nucleic acid sequence
elements to one another, chemically synthesizing selected sequence
elements, and/or otherwise generating a nucleic acid that encodes
and/or directs expression of the polypeptide or one or more
component(s), portion(s), element(s), or domain(s) thereof. In some
embodiments, one or more of such selected sequence elements is
found in nature. In some embodiments, one or more of such selected
sequence elements is designed in silico. In some embodiments, one
or more such selected sequence elements results from mutagenesis
(e.g., in vivo or in vitro) of a known sequence element, e.g., from
a natural or synthetic source such as, for example, in the germline
of a source organism of interest (e.g., of a human, a mouse,
etc.).
[0062] Specific binding: As used herein, the term "specific
binding" refers to an ability to discriminate between possible
binding partners in the environment in which binding is to occur. A
binding agent that interacts with one particular target when other
potential targets are present is said to "bind specifically" to the
target with which it interacts. In some embodiments, specific
binding is assessed by detecting or determining degree of
association between the binding agent and its partner; in some
embodiments, specific binding is assessed by detecting or
determining degree of dissociation of a binding agent-partner
complex; in some embodiments, specific binding is assessed by
detecting or determining ability of the binding agent to compete an
alternative interaction between its partner and another entity. In
some embodiments, specific binding is assessed by performing such
detections or determinations across a range of concentrations.
[0063] Subject: As used herein, the term "subject" refers an
organism, typically a mammal (e.g., a human, in some embodiments
including prenatal human forms). In some embodiments, a subject is
suffering from a relevant disease, disorder or condition. In some
embodiments, a subject is susceptible to a disease, disorder, or
condition. In some embodiments, a subject displays one or more
symptoms or characteristics of a disease, disorder or condition. In
some embodiments, a subject does not display any symptom or
characteristic of a disease, disorder, or condition. In some
embodiments, a subject is someone with one or more features
characteristic of susceptibility to or risk of a disease, disorder,
or condition. In some embodiments, a subject is a patient. In some
embodiments, a subject is an individual to whom diagnosis and/or
therapy is and/or has been administered.
[0064] Therapeutic agent: As used herein, the phrase "therapeutic
agent" in general refers to any agent that elicits a desired
pharmacological effect when administered to an organism. In some
embodiments, an agent is considered to be a therapeutic agent if it
demonstrates a statistically significant effect across an
appropriate population. In some embodiments, the appropriate
population may be a population of model organisms. In some
embodiments, an appropriate population may be defined by various
criteria, such as a certain age group, gender, genetic background,
preexisting clinical conditions, etc. In some embodiments, a
therapeutic agent is a substance that can be used to alleviate,
ameliorate, relieve, inhibit, prevent, delay onset of, reduce
severity of, and/or reduce incidence of one or more symptoms or
features of a disease, disorder, and/or condition. In some
embodiments, a "therapeutic agent" is an agent that has been or is
required to be approved by a government agency before it can be
marketed for administration to humans. In some embodiments, a
"therapeutic agent" is an agent for which a medical prescription is
required for administration to humans.
[0065] Therapeutically Effective Amount: As used herein, the term
"therapeutically effective amount" means an amount that is
sufficient, when administered to a population suffering from or
susceptible to a disease, disorder, and/or condition in accordance
with a therapeutic dosing regimen, to treat the disease, disorder,
and/or condition. In some embodiments, a therapeutically effective
amount is one that reduces the incidence and/or severity of,
stabilizes one or more characteristics of, and/or delays onset of,
one or more symptoms of the disease, disorder, and/or condition.
Those of ordinary skill in the art will appreciate that the term
"therapeutically effective amount" does not in fact require
successful treatment be achieved in a particular individual.
Rather, a therapeutically effective amount may be that amount that
provides a particular desired pharmacological response in a
significant number of subjects when administered to patients in
need of such treatment. For example, in some embodiments, term
"therapeutically effective amount", refers to an amount which, when
administered to an individual in need thereof in the context of
inventive therapy, will block, stabilize, attenuate, or reverse a
cancer-supportive process occurring in said individual, or will
enhance or increase a cancer-suppressive process in said
individual. In the context of cancer treatment, a "therapeutically
effective amount" is an amount which, when administered to an
individual diagnosed with a cancer, will prevent, stabilize,
inhibit, or reduce the further development of cancer in the
individual. A particularly preferred "therapeutically effective
amount" of a composition described herein reverses (in a
therapeutic treatment) the development of a malignancy such as a
pancreatic carcinoma or helps achieve or prolong remission of a
malignancy. A therapeutically effective amount administered to an
individual to treat a cancer in that individual may be the same or
different from a therapeutically effective amount administered to
promote remission or inhibit metastasis. As with most cancer
therapies, the therapeutic methods described herein are not to be
interpreted as, restricted to, or otherwise limited to a "cure" for
cancer; rather the methods of treatment are directed to the use of
the described compositions to "treat" a cancer, i.e., to effect a
desirable or beneficial change in the health of an individual who
has cancer. Such benefits are recognized by skilled healthcare
providers in the field of oncology and include, but are not limited
to, a stabilization of patient condition, a decrease in tumor size
(tumor regression), an improvement in vital functions (e.g.,
improved function of cancerous tissues or organs), a decrease or
inhibition of further metastasis, a decrease in opportunistic
infections, an increased survivability, a decrease in pain,
improved motor function, improved cognitive function, improved
feeling of energy (vitality, decreased malaise), improved feeling
of well-being, restoration of normal appetite, restoration of
healthy weight gain, and combinations thereof. In addition,
regression of a particular tumor in an individual (e.g., as the
result of treatments described herein) may also be assessed by
taking samples of cancer cells from the site of a tumor such as a
pancreatic adenocarcinoma (e.g., over the course of treatment) and
testing the cancer cells for the level of metabolic and signaling
markers to monitor the status of the cancer cells to verify at the
molecular level the regression of the cancer cells to a less
malignant phenotype. For example, tumor regression induced by
employing the methods of this invention would be indicated by
finding a decrease in any of the pro-angiogenic markers discussed
above, an increase in anti-angiogenic markers described herein, the
normalization (i.e., alteration toward a state found in normal
individuals not suffering from cancer) of metabolic pathways,
intercellular signaling pathways, or intracellular signaling
pathways that exhibit abnormal activity in individuals diagnosed
with cancer. Those of ordinary skill in the art will appreciate
that, in some embodiments, a therapeutically effective amount may
be formulated and/or administered in a single dose. In some
embodiments, a therapeutically effective amount may be formulated
and/or administered in a plurality of doses, for example, as part
of a dosing regimen.
[0066] Variant: As used herein in the context of molecules, e.g.,
nucleic acids, proteins, or small molecules, the term "variant"
refers to a molecule that shows significant structural identity
with a reference molecule but differs structurally from the
reference molecule, e.g., in the presence or absence or in the
level of one or more chemical moieties as compared to the reference
entity. In some embodiments, a variant also differs functionally
from its reference molecule. In general, whether a particular
molecule is properly considered to be a "variant" of a reference
molecule is based on its degree of structural identity with the
reference molecule. As will be appreciated by those skilled in the
art, any biological or chemical reference molecule has certain
characteristic structural elements. A variant, by definition, is a
distinct molecule that shares one or more such characteristic
structural elements but differs in at least one aspect from the
reference molecule. To give but a few examples, a polypeptide may
have a characteristic sequence element comprised of a plurality of
amino acids having designated positions relative to one another in
linear or three-dimensional space and/or contributing to a
particular structural motif and/or biological function; a nucleic
acid may have a characteristic sequence element comprised of a
plurality of nucleotide residues having designated positions
relative to on another in linear or three-dimensional space. In
some embodiments, a variant polypeptide or nucleic acid may differ
from a reference polypeptide or nucleic acid as a result of one or
more differences in amino acid or nucleotide sequence. In some
embodiments, a variant polypeptide or nucleic acid shows an overall
sequence identity with a reference polypeptide or nucleic acid that
is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, or 99%. In some embodiments, a variant polypeptide or
nucleic acid does not share at least one characteristic sequence
element with a reference polypeptide or nucleic acid. In some
embodiments, a reference polypeptide or nucleic acid has one or
more biological activities. In some embodiments, a variant
polypeptide or nucleic acid shares one or more of the biological
activities of the reference polypeptide or nucleic acid.
[0067] Vector: as used herein, refers to a nucleic acid molecule
capable of transporting another nucleic acid to which it has been
linked. One type of vector is a "plasmid", which refers to a
circular double stranded DNA loop into which additional DNA
segments may be ligated. Another type of vector is a viral vector,
wherein additional DNA segments may be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) can
be integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "expression vectors." Standard
techniques may be used for recombinant DNA, oligonucleotide
synthesis, and tissue culture and transformation (e.g.,
electroporation, lipofection). Enzymatic reactions and purification
techniques may be performed according to manufacturer's
specifications or as commonly accomplished in the art or as
described herein. The foregoing techniques and procedures may be
generally performed according to conventional methods well known in
the art and as described in various general and more specific
references that are cited and discussed throughout the present
specification. See e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual 2.sup.nd ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated
herein by reference for any purpose.
Programmed Cell Death Protein 1 (PD-1)
[0068] Programmed cell death protein 1 (PD-1) is an inhibitory
receptor that is expressed by all T cells during activation. In
addition to being expressed by conventional T cells, PD-1 is
expressed by regulatory T cells, B cells, natural killer (NK)
cells, and some myeloid cell populations. PD-1 regulates T cell
effector functions during various physiological responses,
including acute and chronic infection, cancer, autoimmunity, and in
immune homeostasis. Further, PD-1 often shows high and sustained
expression levels during antigen encounter, which can occur in the
setting of chronic infections or cancer and can limit protective
immunity.
[0069] In a tumor microenvironment, PD-1 and its ligand, programmed
death ligand 1 (PD-L1) perform a vital role in tumor progression
and survival by escaping tumor neutralizing immune surveillance.
Enhancing T cell activation by blocking the PD-1 and PD-L1
inhibitory pathway has shown beneficial anti-tumor responses and
long-term remissions in a subset of patients with a broad spectrum
of cancers. Therefore, use of an inhibitor that blocks the
interaction of PD-L1 with the PD-1 can help prevent PD-1
stimulation (e.g., on T cells), thereby increasing T cell function
signals and immune cell responses.
[0070] In some embodiments, specific mutations to wild-type PD-1
are introduced to produce PD-1 polypeptide variants, wherein the
specific mutations induce preferential binding to PD-L1. In some
embodiments, a PD-1 polypeptide variant is a soluble PD-1 protein.
In some embodiments, a PD-1 polypeptide variant comprises an
extracellular domain and a transmembrane domain or a fragment
thereof. The transmembrane domain or fragment thereof is described
herein above.
[0071] In some embodiments, a PD-1 polypeptide variant comprises an
amino acid sequence having 95% or more (e.g., 96% or more, 97% or
more, 98% or more, or 99% or more) sequence identity to SEQ ID NO:
4, SEQ ID NO: 6, or SEQ ID NO: 8, wherein the PD-1 polypeptide
variant comprises an extracellular domain that binds specifically
to PD-L1, and a transmembrane domain or a fragment thereof. In some
embodiments, a PD-1 polypeptide variant comprises an amino acid
sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
[0072] In some embodiments, a PD-1 polypeptide variant has
increased binding affinity to a PD-L1 molecule as compared to a
wild-type PD-1 polypeptide. In some embodiments, a polypeptide
variant has a binding affinity (K.sub.D) for a PD-L1 molecule of
about 1.times.10.sup.-8 to 1.times.10.sup.-10 M, preferably of
about 1.times.10.sup.-9 to 1.times.10.sup.-10 M. In some
embodiments, a polypeptide variant has a binding affinity (K.sub.D)
for a PD-L1 molecule of about 1.10.times.10.sup.-9 M, about
1.037.times.10.sup.-9M, or about 7.14.times.10.sup.-10 M.
[0073] As used herein, "nucleic acid" is used to include any
compound and/or substance that comprise polynucleotides. Exemplary
nucleic acids or polynucleotides can include, but are not limited
to, ribonucleic acids (RNAs) and/or deoxyribonucleic acids
(DNAs).
[0074] In some embodiments, a nucleic acid includes a sequence
encoding a PD-1 polypeptide variant, wherein the PD-1 polypeptide
variant comprises at least one of a nucleotide sequence having 95%
or more (96% or more, 97% or more, 98% or more, or 99% or more)
sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:
7.
TABLE-US-00001 human PD-1 wild type (nucleotide sequence) SEQ ID
NO: 1 TTTTTGGATTCTCCAGACCGGCCTTGGAACCCGCCCACGTTTAGCCCTGCT
CTTTTGGTAGTTACAGAGGGGGACAACGCCACATTCACCTGCAGCTTCTCT
AATACGTCCGAGAGCTTTGTACTGAATTGGTATAGAATGAGTCCATCTAAT
CAGACAGATAAATTGGCTGCCTTCCCTGAAGACAGGAGTCAGCCGGGTCAG
GACTGCAGATTCCGCGTTACGCAACTCCCAAATGGTCGAGACTTTCATATG
TCAGTTGTTCGGGCGAGGAGAAATGATAGCGGTACTTACCTGTGCGGCGCG
ATATCTCTCGCACCAAAAGCACAGATTAAAGAGTCTCTCCGGGCTGAACTC
CGCGTGACAGAAAGGCGAGCCGAGGTACCAACGGCGCACCCATCACCGAGT
CCTAGACCTGCGGGCCAATTCCAGACTTTGGTTGTCGGA human PD-1 wild type (amino
acid) SEQ ID NO: 2
FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSN
QTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGA
ISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVG PD-1 polypeptide
variant PD-1.m7 (nucleotide sequence) SEQ ID NO: 3
TTTCTGGAGTCACCCGACCGCCCTTGGAACGCACCTACCTTTTCCCCGGCC
CTCTTGCTGGTCGCAGAAGGAGATAACGCCACTTTCACATGCAGCTTTAGT
AACGCCTCCGAATCTTTTCATGTAGTTTGGCACAGAGAAAGCCCCTCCGGA
CAAACCGACACCTTGGCTGCGTTTCCCGAAGACCGAAGTCAACCAGGGCAG
GACTGCCGGTTCCGCGTAACACGGCTGCCAAACGGTAGGGACTTCCACATG
TCAGTGGTTCGAGCACGCCGGAACGACAGCGGGACGTATGTCTGCGGAGTC
ATTAGCCTTGCCCCGAAGATACAGATTAAAGAAAGTCTTGGGGCAGAACTT
CGAGTCACCGAGCGCAGGGCCGAGGTCCCAACGGCACATCCCAGTCCTAGT
CCACGGCCCGCCGGTCAATTTCAGACCCTTGTAGTGGGC PD-1 polypeptide variant
PD-1.m7 (amino acid) SEQ ID NO: 4
FLESPDRPWNAPTFSPALLLVAEGDNATFTCSFSNASESFHVVWHRESPSG
QTDTLAAFPEDRSQPGQDCRFRVTRLPNGRDFHMSVVRARRNDSGTYVCGV
ISLAPKIQIKESLGAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVG PD-1 polypeptide
variant PD-1.m8 (nucleotide sequence) SEQ ID NO: 5
TTTTTGGAAAGTCCCGATCGGCCTTGGAACGCTCCCACATTTAGCCCGGCC
CTGCTTTTGGTTGCTGAAGGCGATAACGCCACTTTTACATGCAGTTTCAGC
AACGCCTCTGAAAGTTTCCATGTAGTGTGGCACCGCGAGTCTCCAAGTGGG
CAAACAGATACCCTTGCAGCTTTCCCGGAAGATAGGAGTCAGCCAGGGCAG
GATCACCGGTTTAGAGTCACTCGCCTCCCCAATGGTAGAGATTTTCACATG
AGCGTCGTTCGAGCTCAGAGAAACGATAGTGGCACATACGTTTGTGGCGTA
ATATCTCTCGCCCCGAAGATCCAGATTAAAGAGTCCCTTGGCGCGGAGCTG
AGAGTCACCGAGAGGCGAGCTGAGGTGCCTACAGCTCATCCTAGCCCGAGC
CCAAGGCCAGCTGGACAGTTCCAAACTTTGGTTGTAGGC PD-1 polypeptide variant
PD-1.m8 (amino acid) SEQ ID NO: 6
FLESPDRPWNAPTFSPALLLVAEGDNATFTCSFSNASESFHVVWHRESPSG
QTDTLAAFPEDRSQPGQDHRFRVTRLPNGRDFHMSVVRAQRNDSGTYVCGV
ISLAPKIQIKESLGAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVG PD-1 polypeptide
variant euPD-1 (nucleotide sequence) SEQ ID NO: 7
TTTCTCGAATCACCGGACAGACCCTGGAATGCGCCCACATTCTCACCAGCA
CTTTTGCTGGTAGCAGAGGGCGATAATGCTACATTCACGTGTTCCTTCAGT
AATGCAAGCGAGTCATTTCATGTGGTTTGGCATCGAGAGTCACCTAGTGGG
CAGACTGATACACTTGCCGCATTCCCGGAAGATCGCTCCCAGCCAGGTCAG
GATCACCGGTTCAGGGTAACCCGACTGCCGAATGGGCGCGATTTCCATATG
AGCGTTGTCCGGGCGCAACGGAACGATAGTGGAACATACGTGTGTGGCGTA
ATATCCCTCGCTCCCAAAATACAAATAAAGGAGTCTCTGAGAGCAGAGCTG
AGAGTGACAGAACGACGGGCGGAAGTTCCCACGGCTCATCCGTCACCAAGT
CCGCGCCCCGCAGGCCAATTTCAAACGCTCGTCGTAGGC PD-1 polypeptide variant
euPD-1 (amino acid) SEQ ID NO: 8
FLESPDRPWNAPTFSPALLLVAEGDNATFTCSFSNASESFHVVWHRESPSG
QTDTLAAFPEDRSQPGQDHRFRVTRLPNGRDFHMSVVRAQRNDSGTYVCGV
ISLAPKIQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVG
[0075] In all embodiment described herein the PD-1 variant may
include additional residues at the C-terminus arising from
selection of the restriction endonuclease used for cloning. It is
preferred that the additional residues arising from cloning
selection is limited to one, two, three, or four residues with
preference given to a dipeptide addition, preferably an
alanine-serine dipeptide. For example when used as the PD-1
polypeptide variant on its own or as part of a fusion construct, in
the case of euPD-1, included in the present invention is the
sequence of SEQ ID NO: 8 plus a C-terminal AS dipeptide. Such a
sequence is envisioned and embraced by the defined level of amino
acid sequence identity for the PD-1 polypeptide variant of the
present invention.
[0076] In some embodiments, the PD-1 polypeptide variant of the
present invention includes a PD-1 variant comprising an amino acid
sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or
more, or 99% or more) sequence identity to residues 24 to 172 of
SEQ ID NO: 11, wherein said variant has a mutation at least at one
residue selected from the group consisting of D26, P34, V43, T45,
T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125,
A132, and R139. Included in the present invention is nucleic acid
encoding a PD-1 polypeptide variant of this embodiment, a vector
comprising the nucleic acid, a pharmaceutical composition
comprising the PD-1 polypeptide variant of this embodiment, and a
method of treating disease or condition (e.g., cancer) in a subject
in need thereof by administering the pharmaceutical
composition.
[0077] In a variant of this embodiment, the PD-1 polypeptide
variant of the present invention includes a PD-1 variant comprising
an amino acid sequence having 95% or more (e.g., 96% or more, 97%
or more, 98% or more, or 99% or more) sequence identity to residues
24 to 172 of SEQ ID NO: 11, wherein said variant has a mutation at
D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, Q99,
L122, A125, A132, and R139. Included in the present invention is
nucleic acid encoding a PD-1 polypeptide variant of this
embodiment, a vector comprising the nucleic acid, a pharmaceutical
composition comprising the PD-1 polypeptide variant of this
embodiment, and a method of treating disease or condition (e.g.,
cancer) in a subject in need thereof by administering the
pharmaceutical composition.
[0078] In some embodiments, the PD-1 polypeptide variant of the
present invention includes a PD-1 variant comprising an amino acid
sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or
more, or 99% or more) sequence identity to residues 24 to 172 of
SEQ ID NO: 11, wherein said variant has a mutation at D26, P34,
V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114,
L122, A125, A132, and R139. Included in the present invention is
nucleic acid encoding a PD-1 polypeptide variant of this
embodiment, a vector comprising the nucleic acid, a pharmaceutical
composition comprising the PD-1 polypeptide variant of this
embodiment, and a method of treating disease or condition (e.g.,
cancer) in a subject in need thereof by administering the
pharmaceutical composition.
[0079] Preferred mutations include: D26E, P34A, V43L, T45A, T59A,
V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122Y,
A125V, A132I, R139G.
Fc-Fusion Proteins and Encoding Nucleotide Sequence
[0080] As used herein, "Fc-fusion proteins" refer to engineered
proteins composed of the Fc domain of IgG linked to a polypeptide
or protein of interest. Examples of Fc-fused polypeptides or
proteins include, but are not limited to, single peptides, ligands
activated upon cell-surface receptor binding, signaling molecules
(e.g., cytokines), extracellular domains of a receptor activated
upon dimerization, and bait proteins used to identify binding
partners in protein microarrays. In some embodiments, a Fc-fusion
protein acts as an antibody agent that specifically binds to a
particular antigen via an antigen binding domain. In some
embodiments, the Fc fusion protein includes a polypeptide or
polypeptide complex that includes immunoglobulin structural
elements sufficient to confer specific binding.
[0081] Fc is the constant domain selected from an IgG1 or a variant
thereof, an IgG2 or a variant thereof, an IgG4 or a variant
thereof, as well as humanized IgG1/2 or a variant thereof. Fc plays
multiple roles in dimerization for formation of Y-shaped structure
of Ig and maintenance of the structure, and Fc-mediated effector
functions and extension of serum half-life. In some embodiments,
there are two domains, second constant domain (CH2) and third
constant domain (CH3) in monomeric Fc. In some embodiments, the
fusion protein includes a linker that fuses the Fc domain and
specific peptide or protein together. In some embodiments, the
immunoglobulin Fc region is linked to the specific peptide or
protein by a peptide bond. In some embodiments, the immunoglobulin
Fc region is linked to the specific peptide or protein by a peptide
linker sequence. In some embodiments, the linker sequence comprises
a (G.sub.4S).sub.2 (SEQ ID NO: 19), a (G.sub.4S).sub.3 linker (SEQ
ID NO: 20), or a 218 linker (SEQ ID NO: 21).
[0082] In some embodiments, the specific peptide or protein is
linked to the carboxy-terminus of the immunoglobulin Fc region. In
some embodiments, the Fc domain comprises SEQ ID NO: 10 or SEQ ID
NO: 16 or a sequence having 95% or more (e.g., 96% or more, 97% or
more, 98% or more, or 99% or more) sequence identity to SEQ ID NO:
10 or SEQ ID NO: 16.
[0083] In some embodiments, a PD-1 polypeptide variant is used to
produce a PD-1 fusion protein. In some embodiments, a PD-1 fusion
protein can include a PD-1 polypeptide variant and an
immunoglobulin Fc region. In some embodiments, a PD-1 Fc fusion
protein comprises an immunoglobulin Fc region, and a PD-1
polypeptide variant linked by a peptide bond or a peptide linker
sequence to the carboxy-terminus of the immunoglobulin Fc region,
wherein the PD-1 polypeptide variant comprises an amino acid
sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or
more, 99% or more) sequence identity to SEQ ID NO: 4, SEQ ID NO: 6,
or SEQ ID NO: 8.
[0084] In some embodiments, the immunoglobulin Fc region comprises
an amino acid sequence having 95% or more (e.g., 96% or more, 97%
or more, 98% or more, or 99% or more) sequence identity to SEQ ID
NO: 10 or SEQ ID NO: 16.
[0085] In some embodiments, a PD-1 Fc fusion protein has increased
binding affinity to a PD-L1 molecule as compared to a wild-type
PD-1 polypeptide. In some embodiments, a PD-1 Fc fusion protein has
a binding affinity (K.sub.D) for a PD-L1 molecule of about
1.times.10.sup.-8 to 1.times.10.sup.-10 M, preferably of about
1.times.10.sup.-9 to 1.times.10.sup.-10 M. In some embodiments, a
PD-1 Fc fusion protein has a binding affinity (KD) for a PD-L1
molecule of about 1.10.times.10.sup.-9 M, about
1.037.times.10.sup.-9M, or about 7.14.times.10.sup.-10 M.
[0086] As used herein, "nucleic acid" is used to include any
compound and/or substance that comprise polynucleotides. Exemplary
nucleic acids or polynucleotides can include, but are not limited
to, ribonucleic acids (RNAs) and/or deoxyribonucleic acids
(DNAs).
[0087] In some embodiments, nucleic acid constructs include regions
that encode an immunoglobulin Fc region and a PD-1 polypeptide
variant. In some embodiments, the sequence encoding the Fc domain
comprises nucleotide sequence having 95% or more (e.g., 95% or
more, 96% or more, 97% or more, 98% or more, or 99% or more)
sequence identity to SEQ ID NO: 9.
TABLE-US-00002 Fc (207-230 IMGT allele IGHG1*03, 231-457 IMGT
allele IGHG2*01) SEQ ID NO: 9
AGCAACACTAAAGTCGACAAGCGAGTAGAACCGAAATCATGCGACAAAACA
CATACGTGCCCTCCCTGCCCAGCACCACCTGTCGCGGGCCCCTCTGTTTTC
CTGTTTCCACCCAAGCCAAAGGACACATTGATGATTTCCCGGACTCCTGAA
GTCACCTGCGTGGTAGTAGATGTATCACATGAAGATCCAGAAGTCCAGTTC
AACTGGTATGTGGACGGAGTAGAGGTACATAATGCCAAGACCAAACCACGG
GAAGAGCAGTTCAACAGTACTTTCCGGGTAGTTAGCGTTTTGACTGTCGTA
CACCAAGACTGGCTTAATGGAAAAGAATACAAGTGTAAGGTAAGCAACAAG
GGCCTGCCGGCTCCGATAGAGAAAACCATTAGCAAGACAAAGGGCCAACCA
CGCGAACCCCAGGTATATACCCTCCCACCGTCCCGCGAGGAGATGACTAAG
AATCAAGTTTCTCTCACGTGCTTGGTAAAGGGCTTCTATCCGAGCGATATA
GCCGTGGAGTGGGAGTCTAATGGTCAGCCCGAAAACAATTACAAAACTACG
CCTCCTATGCTGGACAGTGATGGGAGCTTCTTTCTTTACAGTAAGCTTACC
GTGGACAAGTCTCGGTGGCAACAAGGAAATGTTTTTAGTTGTTCTGTAATG
CATGAAGCACTTCATAACCATTACACCCAGAAAAGTCTGAGCTTGTCCCCG GGAAAA Fc
(207-230 IMGT allele IGHG1*03, 231-457 IMGT allele IGHG2*01) (amino
acid) SEQ ID NO: 10
SNTKVDKRVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVV
HQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0088] As explained above, in all embodiment described herein the
PD-1 variant may include additional residues at the C-terminus
arising from selection of the restriction endonuclease used for
cloning. It is preferred that the additional residues arising from
cloning selection is limited to one, two, three, or four residues
with preference given to a dipeptide addition, preferably an
alanine-serine dipeptide. For example when used as the PD-1
polypeptide variant on its own or as part of a fusion construct, in
the case of euPD-1, included in the present invention is the
sequence of SEQ ID NO: 8 plus a C-terminal AS dipeptide. Such a
sequence is envisioned and embraced by the defined level of amino
acid sequence identity for the PD-1 polypeptide variant of the
present invention. Thus, in a fusion construct of this embodiment
the residues arising from cloning may be located before the
linker.
[0089] Also embraced by the present invention is a PD-1 Fc fusion
protein wherein multiple copies of the PD-1 polypeptide variant is
present, each separated by a linker. The term "multiple copies of
the PD-1 polypeptide variant" means that two, three, or four PD-1
polypeptide variants may be present wherein the variants present
may be the same or different. In addition, the linker separating
the respective PD-1 polypeptide variants may be the same or
different. Also, the linker between the PD-1 polypeptide variants
may be different from the linker between the PD-1 polypeptide
variant and the polypeptide or protein of interest.
[0090] In some embodiments, the PD-1 polypeptide variant for the
PD-1 Fc fusion protein comprises an amino acid sequence having 95%
or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or
more) sequence identity to residues 24 to 172 of SEQ ID NO: 11,
wherein said variant has a mutation at least at one residue
selected from the group consisting of D26, P34, V43, T45, T59, V64,
L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and
R139. Included in the present invention is nucleic acid encoding a
PD-1 polypeptide variant of this embodiment, a vector comprising
the nucleic acid, a pharmaceutical composition comprising the PD-1
polypeptide variant of this embodiment, and a method of treating
disease or condition (e.g., cancer) in a subject in need thereof by
administering the pharmaceutical composition. In a variant of this
embodiment, the PD-1 polypeptide variant for the PD-1 Fc fusion
protein comprises an amino acid sequence having 95% or more (e.g.,
96% or more, 97% or more, 98% or more, or 99% or more) sequence
identity to residues 24 to 172 of SEQ ID NO: 11, wherein said
variant has a mutation at D26, P34, V43, T45, T59, V64, L65, N66,
Y68, M70, N74, K78, Q99, L122, A125, A132, and R139. Included in
the present invention is nucleic acid encoding a PD-1 polypeptide
variant of this embodiment, a vector comprising the nucleic acid, a
pharmaceutical composition comprising the PD-1 polypeptide variant
of this embodiment, and a method of treating disease or condition
(e.g., cancer) in a subject in need thereof by administering the
pharmaceutical composition.
[0091] In some embodiments, the PD-1 polypeptide variant for the
PD-1 Fc fusion protein comprises an amino acid sequence having 95%
or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or
more) sequence identity to residues 24 to 172 of SEQ ID NO: 11,
wherein said variant has a mutation at D26, P34, V43, T45, T59,
V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125,
A132, and R139. Included in the present invention is nucleic acid
encoding a PD-1 polypeptide variant of this embodiment, a vector
comprising the nucleic acid, a pharmaceutical composition
comprising the PD-1 polypeptide variant of this embodiment, and a
method of treating disease or condition (e.g., cancer) in a subject
in need thereof by administering the pharmaceutical
composition.
[0092] Preferred mutations include: D26E, P34A, V43L, T45A, T59A,
V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122Y,
A125V, A132I, R139G.
[0093] As stated above, in some embodiments, the immunoglobulin Fc
region comprises an amino acid sequence having 95% or more (e.g.,
96% or more, 97% or more, 98% or more, or 99% or more) sequence
identity to SEQ ID NO: 10 or SEQ ID NO: 16.
4-1BB
[0094] 4-1BB (also referred to as CD137, TNFRSF9) is a receptor
belonging to the tumor necrosis factor receptor (TNFR) superfamily.
4-1BB is a co-stimulatory molecule generally expressed in activated
T lymphocytes and involved in immunity and autoimmune diseases
(Kwon et al. PNAS 84: 2896, 1987; Kwon et al. PNAS 86: 1963, 1989;
Son et al. Journal of Immunological Methods 286 (1-2): 187-201,
2004, each of which is herein incorporated by reference in its
entirety). Human 4-1BB is a 255 amino acid protein and expressed on
the cell surface in monomer (30 kDa) and dimer (55 kDa) forms and
likely trimerizes with 4-1BB ligand to signal.
[0095] Further, 4-1BB is constitutively expressed on a number of
cells, albeit at low levels, including Foxp3+ Tregs and dendritic
cells (DC). Activation with a number of agonists, such as cytokines
(e.g., IL-2, IL-4), polyclonal activators (e.g., Con A and PHA),
cell surface molecules (e.g., anti-CD3, anti-CD28) and promoters of
Ca.sup.2+ induction and PKC activity (e.g., ionomycin, photbol
myristate acetate) further enhance expression of 4-1BB.
[0096] Numerous studies of murine and human T cells indicate that
4-1BB promotes enhanced cellular proliferation, survival, and
cytokine production. Studies have indicated that some 4-1BB agonist
monoclonal antibodies can increase costimulatory molecule
expression and markedly enhance cytolytic T lymphocyte responses,
resulting in anti-tumor efficacy in prophylactic and therapeutic
settings. Further, 4-1BB monotherapy and combination therapy tumor
models have established durable anti-tumor protective T cell memory
responses. 4-1BB agonists also have been shown to inhibit
autoimmune reactions in a variety of art-recognized autoimmunity
models. This dual activity of 4-1BB offers the potential to provide
anti-tumor activity while dampening autoimmune side effects that
can be associated with immunotherapy approaches.
[0097] In some embodiments herein, the fusion protein may contain
as the immunoglobulin Fc region an anti-4-1BB antibody domain.
Specifically, an anti-4-1BB antibody domain can be produced using
94kvt clones (WO2018-127787, incorporated herein by reference in
its entirety) possessing an anti-4-1BB antibody domain (94kvt) as a
single chain Fv (scFv). Examples of suitable scFvs for the
anti-4-1BB antibody, VH-218 linker-VL (HLC 218) and VL-218
linker-VH (LHC 218). The 94kvt VH sequence is shown in SEQ ID NO:
17, while the 94kvt VL sequence is shown in SEQ NO: 18 and the 218
liker is shown in SEQ ID NO: 21. Further, the scFv variant 94kvt
HLC 218 is shown in SEQ ID NO: 22 and the scFv variant 94kvt LHC
218 is shown in SEQ ID NO: 23.
[0098] In some embodiments, the anti-4-1BB antibody domain (94kvt)
as a single chain Fv (scFv) includes a sequence that is at least
70% identical (e.g., at least 75% identical, at least 80%
identical, at least 85% identical, at least 90% identical, at least
95% identical, at least 96% identical, at least 97% identical, at
least 98% identical, at least 99% identical, or 100% identical) to
SEQ ID NO: 22 or SEQ ID NO: 23.
[0099] Thus, in an embodiment of the present invention is a Fc
fusion BsAb (Bispecific antibody) comprising; an immunoglobulin Fc
region; and a PD-1 polypeptide variant linked by a peptide bond or
a peptide linker sequence to the N-terminus of the immunoglobulin
Fc region, wherein the PD-1 polypeptide variant comprises an amino
acid sequence having 95% or more (e.g., 96% or more, 97% or more,
98% or more, 99% or more) sequence identity to SEQ ID NO: 4, SEQ ID
NO: 6, or SEQ ID NO: 8; and a scFv for the anti-4-1BB antibody
linked by a peptide bond or a peptide linker sequence to the
C-terminus of the immunoglobulin Fc region, wherein the scFv for
the anti-4-1BB antibody comprises an amino acid sequence having 95%
or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more)
sequence identity to an amino acid sequence comprising SEQ ID NO:
17 and 18 linked by a peptide bond or a peptide linker
sequence.
[0100] Also provided is a Fc fusion BsAb (Bispecific antibody)
comprising; an immunoglobulin Fc region; and a PD-1 polypeptide
variant linked by a peptide bond or a peptide linker sequence to
the N-terminus of the immunoglobulin Fc region, wherein the PD-1
polypeptide variant comprises an amino acid sequence having 95% or
more (e.g., 96% or more, 97% or more, 98% or more, 99% or more)
sequence identity to residues 24 to 172 of SEQ ID NO: 11, wherein
said variant has a mutation at least at one residue selected from
the group consisting of D26, P34, V43, T45, T59, V64, L65, N66,
Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139; and
a scFv for the anti-4-1BB antibody linked by a peptide bond or a
peptide linker sequence to the C-terminus of the immunoglobulin Fc
region, wherein the scFv for the anti-4-1BB antibody comprises an
amino acid sequence having 95% or more (e.g., 96% or more, 97% or
more, 98% or more, 99% or more) sequence identity to an amino acid
sequence comprising SEQ ID NO: 17 and 18 linked by a peptide bond
or a peptide linker sequence.
[0101] In some embodiments, the immunoglobulin Fc region comprises
an amino acid sequence having 95% or more (e.g., 96% or more, 97%
or more, 98% or more, or 99% or more) sequence identity to SEQ ID
NO: 10 or SEQ ID NO: 16.
[0102] In some embodiments the scFv for the anti-4-1BB antibody is
an amino acid sequence that is at least 70% identical (e.g., at
least 75% identical, at least 80% identical, at least 85%
identical, at least 90% identical, at least 95% identical, at least
96% identical, at least 97% identical, at least 98% identical, at
least 99% identical, or 100% identical) to SEQ ID NO: 22 or SEQ ID
NO: 23.
[0103] Examples of the linker sequence include a (G.sub.4S).sub.2
(SEQ ID NO: 19), a (G.sub.4S).sub.3 linker (SEQ ID NO: 20), or a
218 linker (SEQ ID NO: 21).
[0104] The bispecific antibody described herein has increased
binding affinity to a PD-L1 molecule as compared to a wild-type
PD-1 polypeptide and specific binding affinity to 4-1BB.
[0105] The BsAb antibody of the present invention has decreased or
no antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC) effects.
[0106] The BsAb antibody of the present invention demonstrates
decreased or no hepatotoxicity.
Vectors
[0107] In some embodiments, nucleic acid constructs described above
may be inserted into an expression vector or viral vector by
methods known to the art, and nucleic acid molecules may be
operably linked to an expression control sequence. Non-limiting
examples of expression vectors include plasmid vectors, transposon
vectors, cosmid vectors, and viral derived vectors (e.g., any
adenoviral derived vectors (AV), cytomegaloviral derived (CMV)
vectors, simian viral derived (SV40) vectors, adeno-associated
virus (AAV) vectors, lentivirus vectors, and retroviral vectors).
In some embodiments, the expression vector is a viral vector.
[0108] Additional sequences can be added to such cloning and/or
expression sequences to optimize their function in cloning and/or
expression, to aid in isolation of the polynucleotide, or to
improve the introduction of the polynucleotide into a cell. Use of
cloning vectors, expression vectors, adapters, and linkers is well
known in the art.
[0109] In some embodiments, nucleic acid molecules are inserted
into a vector that is able to express a PD-1 polypeptide variant of
the present disclosure or a PD-1 Fc fusion protein when introduced
into an appropriate cell.
Therapeutic Applications
[0110] In some embodiments, the PD-1 polypeptide variants, PD-1 Fc
fusion proteins, or nucleic acid constructs described herein may be
used for treating a subject in need thereof. In some embodiments,
the subject is diagnosed with a PD-L1 associated disease. In some
embodiments, the subject is diagnosed with a PD-L1 associated
cancer. In some embodiments, a pharmaceutical composition that
includes a PD-1 polypeptide variant or a PD-1 Fc fusion protein,
and a pharmaceutically acceptable carrier can be administered to
the subject diagnosed with a PD-L1 associated disease. In some
embodiments, the pharmaceutical composition can be administered
with one or more additional anticancer therapies that include, but
are not limited to, ionizing radiation, a chemotherapeutic agent, a
therapeutic antibody, and a checkpoint inhibitor.
[0111] The PD-1/PD-L1 pathway represents an adaptive immune
resistance mechanism used by tumor cells in response to endogenous
immune anti-tumor activity. PD-L1 expressed on tumor cells binds to
PD-1 receptors on activated T cells, which leads to the inhibition
of the cytotoxic T cells. In some embodiments, PD-1 polypeptide
variants are used as PD-L1 inhibitors to treat cancers that can
include, but are not limited to, non-small cell lung cancer, lung
adenocarcinoma, gastric cancer, and breast cancer.
[0112] Cancer can refer to a broad group of diseases characterized
by the uncontrolled growth of abnormal cells in the body.
Unregulated cell division and growth results in the formation of
malignant tumors that invade neighboring tissues and may also
metastasize to distant parts of the body through the lymphatic
system or bloodstream. Cancer or cancer tissue may include a
tumor.
[0113] Cancers suitable for treatment by a method of the present
disclosure can include, but are not limited to, bladder cancer,
breast cancer, cervical cancer, colon cancer, endometrial cancer,
esophageal cancer, fallopian tube cancer, gall bladder cancer,
gastrointestinal cancer, head and neck cancer, hematological
cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma,
melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer,
salivary gland cancer, sarcoma, stomach cancer, thyroid cancer,
pancreatic cancer, and prostate cancer. In some embodiments, a
cancer for treatment by a method of the present disclosure can
include may include, but is not limited to, carcinoma, lymphoma
(e.g., Hodgkin's and non-Hodgkin's lymphomas), blastoma, sarcoma,
and leukemia. In some embodiments, cancer may include squamous cell
carcinoma, small cell lung cancer, non-small cell lung cancer, lung
adenocarcinoma, squamous cell carcinoma of the lung, peritoneal
cancer, hepatocellular carcinoma, gastric cancer, pancreatic
cancer, glioma, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, hepatocellular carcinoma, breast cancer, colon
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary carcinoma, kidney cancer, prostate cancer, vulvar cancer,
thyroid cancer, liver carcinoma, leukemia and other
lymphoproliferative disorders, and various types of head and neck
cancer.
[0114] In some embodiments, the cancer can be an embryonal tumor
(Wilms tumor, hepatoblastoma, rhabdoid, neuroblasoma), germ cell
tumor (yolk sac tumor, immature teratoma, and embryonal carcinoma),
carcinoma (hepatocellular carcinoma and pulmonary squamous cell
carcinoma), sarcoma (malignant rhabdoid tumor and RMS), or
malignant melanoma.
[0115] The fusion proteins and bispecific antibody of the present
invention have the increased advantage that they have decreased or
no antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC) effects. Further, fusion
proteins and bispecific antibody of the present invention have
decreased or no hepatotoxicity.
[0116] In the context of the present description, all publications,
patent applications, patents and other references mentioned herein,
if not otherwise indicated, are explicitly incorporated by
reference herein in their entirety for all purposes as if fully set
forth, and shall be considered part of the present disclosure in
their entirety.
[0117] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs. In case
of conflict, the present specification, including definitions, will
control.
[0118] The above written description of the invention provides a
manner and process of making and using it such that any person
skilled in this art is enabled to make and use the same, this
enablement being provided in particular for the subject matter of
the appended claims, which make up a part of the original
description.
[0119] As used herein, the phrases "selected from the group
consisting of," "chosen from," and the like include mixtures of the
specified materials.
[0120] Where a numerical limit or range is stated herein, the
endpoints are included. Also, all values and subranges within a
numerical limit or range are specifically included as if explicitly
written out.
[0121] The above description is presented to enable a person
skilled in the art to make and use the invention, and is provided
in the context of a particular application and its requirements.
Various modifications to the preferred embodiments will be readily
apparent to those skilled in the art, and the generic principles
defined herein may be applied to other embodiments and applications
without departing from the spirit and scope of the invention. Thus,
this invention is not intended to be limited to the embodiments
shown, but is to be accorded the widest scope consistent with the
principles and features disclosed herein.
[0122] Without being bound to the following specific embodiments,
the present invention is exemplified by the follow:
[0123] (1) A programmed cell death 1 (PD-1) polypeptide variant
comprising an amino acid sequence having 95% or more (e.g., 96% or
more, 97% or more, 98% or more, or 99% or more) sequence identity
to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8,
[0124] wherein the PD-1 polypeptide variant comprises:
[0125] an extracellular domain that binds specifically to
programmed cell death 1 ligand (PD-L1); and
[0126] a transmembrane domain or a fragment thereof.
[0127] (2) The PD-1 polypeptide variant of (1), wherein the PD-1
polypeptide variant comprises an amino acid sequence having 97% or
more sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO:
8.
[0128] (3) The PD-1 polypeptide variant of any one of (1) or (2),
wherein the PD-1 polypeptide variant comprises an amino acid
sequence having 98% or more sequence identity to SEQ ID NO: 4, SEQ
ID NO: 6, or SEQ ID NO: 8.
[0129] (4) The PD-1 polypeptide variant any one of (1) to (3),
wherein the PD-1 polypeptide variant comprises an amino acid
sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
[0130] (5) The PD-1 polypeptide variant of (1), wherein the PD-1
polypeptide variant comprises SEQ ID NO: 4.
[0131] (6) The PD-1 polypeptide variant of (1), wherein the PD-1
polypeptide variant comprises SEQ ID NO: 6.
[0132] (7) The PD-1 polypeptide variant of (1), wherein the PD-1
polypeptide variant comprises SEQ ID NO: 8.
[0133] (8) The PD-1 polypeptide variant of any one of (1) to (7),
wherein the transmembrane domain comprises at least two amino acid
residues.
[0134] (9) The PD-1 polypeptide variant of any one of (1) to (8),
wherein the transmembrane domain comprises at least 5 amino acid
residues.
[0135] (10) The PD-1 polypeptide variant of any one of (1) to (9),
wherein the transmembrane domain comprises at least 10 amino acid
residues.
[0136] (11) The PD-1 polypeptide variant of any one of (1) to (10),
wherein the polypeptide variant has increased binding affinity to a
PD-L1 molecule as compared to a wild-type PD-1 polypeptide.
[0137] (12) The PD-1 polypeptide variant of any one of (1) to (11),
wherein the polypeptide variant has a binding affinity (KD) for a
PD-L1 molecule of about 1.times.10.sup.-8 to 1.times.10.sup.-10
M.
[0138] (13) The PD-1 polypeptide variant of any one of (1) to (12),
wherein the polypeptide variant has a binding affinity (KD) for a
PD-L1 molecule of about 1.10.times.10.sup.-9M, about
1.037.times.10.sup.-9M, or about 7.14.times.10.sup.-10 M.
[0139] (14) A PD-1 Fc fusion protein comprising:
[0140] an immunoglobulin Fc region; and
[0141] a PD-1 polypeptide variant linked by a peptide bond or a
peptide linker sequence to the carboxy-terminus of the
immunoglobulin Fc region, wherein the PD-1 polypeptide variant
comprises an amino acid sequence having 95% or more (e.g., 96% or
more, 97% or more, 98% or more, or 99% or more) sequence identity
to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
[0142] (15) The PD-1 Fc fusion protein of (14), wherein the
immunoglobulin Fc region comprises an amino acid sequence having
95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or
more) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.
[0143] (16) The PD-1 Fc fusion protein of any one of (14) to (15),
wherein the fusion protein has increased binding affinity to a
PD-L1 molecule as compared to a wild-type PD-1 polypeptide.
[0144] (17) The PD-1 Fc fusion protein of any one of (14) to (16),
wherein the fusion protein has a binding affinity (KD) for a PD-L1
molecule of about 1.times.10.sup.-8 to 1.times.10.sup.-10 M.
[0145] (18) The PD-1 Fc fusion protein of any one of (14) to (17),
wherein the fusion protein has a binding affinity (KD) for a PD-L1
molecule of about 1.10.times.10.sup.-9 M, about
1.037.times.10.sup.-9 M, or about 7.14.times.10.sup.-10 M.
[0146] (19) The PD-1 Fc fusion protein of any one of (14) to (18),
wherein the PD-1 polypeptide variant is linked by a peptide linker
sequence to the carboxy-terminus of the immunoglobulin Fc
region.
[0147] (20) The PD-1 Fc fusion protein of any one of (14) to (19),
wherein the peptide linker sequence is selected from the group
consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
[0148] (21) The PD-1 Fc fusion protein of any one of (14) to (20),
wherein the more than one copy of the PD-1 polypeptide variant is
present wherein the copies may be the same or different and are
linked by a peptide linker sequence.
[0149] (22) The PD-1 Fc fusion protein of (21), wherein two copies
of PD-1 polypeptide variant is present.
[0150] (23) The PD-1 Fc fusion protein of any one of (21) or (22),
wherein the peptide linker sequence is selected from the group
consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
[0151] (24) The PD-1 Fc fusion protein of any one of (14) to (23),
wherein the PD-1 Fc fusion protein is selected from the group
consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO; 14, and SEQ
ID NO: 15.
[0152] (25) A nucleic acid comprising:
[0153] a sequence encoding a PD-1 polypeptide variant, wherein the
polypeptide variant comprises a sequence having 95% or more (e.g.,
96% or more, 97% or more, 98% or more, or 99% or more) sequence
identity to SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.
[0154] (26) The nucleic acid of (25), further comprising:
[0155] a sequence encoding an immunoglobulin Fc region, wherein the
sequence encoding the immunoglobulin Fc region comprises SEQ ID NO:
9.
[0156] (27) An expression vector comprising the nucleic acid of any
one of (25) or (26).
[0157] (28) The vector of (27), wherein the vector is a viral
vector.
[0158] (29) A pharmaceutical composition comprising:
[0159] the PD-1 polypeptide variant of any one of (1) to (13) or
the PD-1 fusion protein of any one of (14) to (24); and
[0160] a pharmaceutically acceptable carrier.
[0161] (30) A method of treating a disease or a condition in a
subject in need thereof, the method comprising:
[0162] administering to the subject the pharmaceutical composition
of (29), thereby treating a disease or a condition.
[0163] (31) The method of (30), wherein the subject has cancer.
[0164] (32) The method of (31), wherein the cancer is selected from
a bladder cancer, breast cancer, cervical cancer, colon cancer,
endometrial cancer, esophageal cancer, fallopian tube cancer, gall
bladder cancer, gastrointestinal cancer, head and neck cancer,
hematological cancer, laryngeal cancer, liver cancer, lung cancer,
lymphoma, melanoma, mesothelioma, ovarian cancer, primary
peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer,
thyroid cancer, pancreatic cancer, renal cell carcinoma,
glioblastoma, and prostate cancer.
[0165] (33) The method of any of (30) to (32), wherein when the
pharmaceutical composition comprises the PD-1 fusion protein of any
one of (14) to (24), decreased or no antibody-dependent cellular
cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC)
effects are observed.
[0166] (34) The method of any of (30) to (33), wherein when the
pharmaceutical composition comprises the PD-1 fusion protein of any
one of (14) to (24), decreased or no hepatotoxicity is
observed.
[0167] (35) A programmed cell death 1 (PD-1) polypeptide variant
comprising an amino acid sequence having 95% or more (e.g., 96% or
more, 97% or more, 98% or more, or 99% or more) sequence identity
to residues 24 to 172 of SEQ ID NO: 11, wherein said variant has a
mutation at least at one residue selected from the group consisting
of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93,
Q99, R114, L122, A125, A132, and R139.
[0168] (36) The PD-1 polypeptide variant of (35), wherein said
variant comprises a mutation at D26, P34, V43, T45, T59, V64, L65,
N66, Y68, M70, N74, K78, Q99, L122, A125, A132, and R139.
[0169] (37) The PD-1 polypeptide variant of (35), wherein said
variant comprises a mutation at D26, P34, V43, T45, T59, V64, L65,
N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and
R139.
[0170] (38) The PD-1 polypeptide variant of any one of (35) to
(37), wherein said mutations are D26E, P34A, V43L, T45A, T59A,
V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122Y,
A125V, A132I, or R139G.
[0171] (39) The PD-1 polypeptide variant of any one of (35) to
(38), wherein the PD-1 polypeptide variant comprises an amino acid
sequence having 97% or more sequence identity to identity to
residues 24 to 172 of SEQ ID NO: 11, wherein said variant has a
mutation at least at one residue selected from the group consisting
of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93,
Q99, R114, L122, A125, A132, and R139.
[0172] (40) The PD-1 polypeptide variant of any one of (35) to
(39), wherein the PD-1 polypeptide variant comprises an amino acid
sequence having 98% or more sequence identity to identity to
residues 24 to 172 of SEQ ID NO: 11, wherein said variant has a
mutation at least at one residue selected from the group consisting
of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93,
Q99, R114, L122, A125, A132, and R139.
[0173] (41) The PD-1 polypeptide variant of any one of (35) to
(40), wherein the polypeptide variant has increased binding
affinity to a PD-L1 molecule as compared to a wild-type PD-1
polypeptide.
[0174] (42) The PD-1 polypeptide variant of any one of (35) to
(41), wherein the polypeptide variant has a binding affinity (KD)
for a PD-L1 molecule of about 1.times.10.sup.-8 to
1.times.10.sup.-10 M.
[0175] (43) A PD-1 Fc fusion protein comprising:
[0176] an immunoglobulin Fc region; and
[0177] a PD-1 polypeptide variant of any one of (35) to (42) linked
by a peptide bond or a peptide linker sequence to the
carboxy-terminus of the immunoglobulin Fc region.
[0178] (44) The PD-1 Fc fusion protein of (43), wherein the
immunoglobulin Fc region comprises an amino acid sequence having
95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or
more) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.
[0179] (45) The PD-1 Fc fusion protein of any one of (43) or (44),
wherein the fusion protein has increased binding affinity to a
PD-L1 molecule as compared to a wild-type PD-1 polypeptide.
[0180] (46) The PD-1 Fc fusion protein of any one of (43) to (45)
wherein the fusion protein has a binding affinity (KD) for a PD-L1
molecule of about 1.times.10.sup.-8 to 1.times.10.sup.-10 M.
[0181] (47) The PD-1 Fc fusion protein of any one of (43) to (46),
wherein the PD-1 polypeptide variant is linked by a peptide linker
sequence to the carboxy-terminus of the immunoglobulin Fc
region.
[0182] (48) The PD-1 Fc fusion protein of any one of (43) to (47),
wherein the peptide linker sequence is selected from the group
consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
[0183] (49) The PD-1 Fc fusion protein of any one of (43) to (48),
wherein the more than one copy of the PD-1 polypeptide variant is
present wherein the copies may be the same or different and are
linked by a peptide linker sequence.
[0184] (50) The PD-1 Fc fusion protein of (49), wherein two copies
of PD-1 polypeptide variant is present.
[0185] (51) The PD-1 Fc fusion protein of any one of (49) or (50),
wherein the peptide linker sequence is selected from the group
consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
[0186] (52) A nucleic acid comprising:
[0187] a sequence encoding a PD-1 polypeptide variant of any one of
(35) to (51).
[0188] (53) The nucleic acid of (52), further comprising:
[0189] a sequence encoding an immunoglobulin Fc region.
[0190] (54) An expression vector comprising the nucleic acid of any
one of (52) or (53).
[0191] (55) The vector of (54), wherein the vector is a viral
vector.
[0192] (56) A pharmaceutical composition comprising:
[0193] the PD-1 polypeptide variant of any one of (35) to (42) or
the PD-1 fusion protein of any one of (43) to (51); and
[0194] a pharmaceutically acceptable carrier.
[0195] (57) A method of treating a disease or a condition in a
subject in need thereof, the method comprising:
[0196] administering to the subject the pharmaceutical composition
of (56), thereby treating a disease or a condition.
[0197] (58) The method of (57), wherein the subject has cancer.
[0198] (59) The method of (58), wherein the cancer is selected from
a bladder cancer, breast cancer, cervical cancer, colon cancer,
endometrial cancer, esophageal cancer, fallopian tube cancer, gall
bladder cancer, gastrointestinal cancer, head and neck cancer,
hematological cancer, laryngeal cancer, liver cancer, lung cancer,
lymphoma, melanoma, mesothelioma, ovarian cancer, primary
peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer,
thyroid cancer, pancreatic cancer, renal cell carcinoma,
glioblastoma, and prostate cancer.
[0199] (60) The method of any of (57) to (59), wherein when the
pharmaceutical composition comprises the PD-1 fusion protein of any
one of (43) to (51), decreased or no antibody-dependent cellular
cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC)
effects are observed.
[0200] (61) The method of any of (57) to (60), wherein when the
pharmaceutical composition comprises the PD-1 fusion protein of any
one of (43) to (51), decreased or no hepatotoxicity is
observed.
[0201] (62) A bispecific antibody comprising:
[0202] an immunoglobulin Fc region;
[0203] a PD-1 polypeptide variant linked by a peptide bond or a
peptide linker sequence to the N-terminus of the immunoglobulin Fc
region, wherein the PD-1 polypeptide variant comprises an amino
acid sequence having 95% or more (e.g., 96% or more, 97% or more,
98% or more, or 99% or more) sequence identity to SEQ ID NO: 4, SEQ
ID NO: 6, or SEQ ID NO: 8; and
[0204] a scFv for the anti-4-1BB antibody linked by a peptide bond
or a peptide linker sequence to the C-terminus of the
immunoglobulin Fc region, wherein the scFv for the anti-4-1BB
antibody comprises an amino acid sequence having 95% or more (e.g.,
96% or more, 97% or more, 98% or more, or 99% or more) sequence
identity to an amino acid sequence comprising SEQ ID NO: 17 and 18
linked by a peptide bond or a peptide linker sequence.
[0205] (63) The bispecific antibody of (62), wherein the
immunoglobulin Fc region comprises an amino acid sequence having
95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or
more) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.
[0206] (64) The bispecific antibody of any of (62) or (63), wherein
the PD-1 polypeptide variant is linked by a peptide linker sequence
to the N-terminus of the immunoglobulin Fc region.
[0207] (65) The bispecific antibody of any of (62) to (64), wherein
the scFv for the anti-4-1BB antibody is linked by peptide linker
sequence to the C-terminus of the immunoglobulin Fc region.
[0208] (66) The bispecific antibody of any of (62) to (65), wherein
the peptide linker sequence is selected from the group consisting
of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
[0209] (67) The bispecific antibody of any of (62) to (66), wherein
the scFv for the anti-4-1BB antibody comprises SEQ ID NO: 22 or SEQ
ID NO: 23 or a sequence that is at least 70% identical (e.g., at
least 75% identical, at least 80% identical, at least 85%
identical, at least 90% identical, at least 95% identical, at least
96% identical, at least 97% identical, at least 98% identical, at
least 99% identical, or 100% identical) to SEQ ID NO: 22 or SEQ ID
NO: 23.
[0210] (68) The bispecific antibody of any of (62) to (67), wherein
the bispecific antibody has increased binding affinity to a PD-L1
molecule as compared to a wild-type PD-1 polypeptide and specific
binding affinity to 4-1BB.
[0211] (69) A pharmaceutical composition comprising:
[0212] the bispecific antibody of any of (62) to (68); and
[0213] a pharmaceutically acceptable carrier.
[0214] (70) A method of treating a disease or a condition in a
subject in need thereof, the method comprising:
[0215] administering to the subject the pharmaceutical composition
of (69), thereby treating a disease or a condition.
[0216] (71) The method of (70), wherein the subject has cancer.
[0217] (72) The method of (71), wherein the cancer is selected from
a bladder cancer, breast cancer, cervical cancer, colon cancer,
endometrial cancer, esophageal cancer, fallopian tube cancer, gall
bladder cancer, gastrointestinal cancer, head and neck cancer,
hematological cancer, laryngeal cancer, liver cancer, lung cancer,
lymphoma, melanoma, mesothelioma, ovarian cancer, primary
peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer,
thyroid cancer, pancreatic cancer, renal cell carcinoma,
glioblastoma, and prostate cancer.
[0218] (73) The method of any of (70) to (72), wherein decreased or
no antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC) effects are observed.
[0219] (74) The method of any of (70) to (73), wherein decreased or
no hepatotoxicity is observed.
[0220] (75) A bispecific antibody comprising:
[0221] an immunoglobulin Fc region;
[0222] a PD-1 polypeptide variant linked by a peptide bond or a
peptide linker sequence to the N-terminus of the immunoglobulin Fc
region, wherein the PD-1 polypeptide variant comprises an amino
acid sequence having 95% or more (e.g., 96% or more, 97% or more,
98% or more, 99% or more) sequence identity to residues 24 to 172
of SEQ ID NO: 11, wherein said variant has a mutation at least at
one residue selected from the group consisting of D26, P34, V43,
T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122,
A125, A132, and R139; and
[0223] a scFv for the anti-4-1BB antibody linked by a peptide bond
or a peptide linker sequence to the C-terminus of the
immunoglobulin Fc region, wherein the scFv for the anti-4-1BB
antibody comprises an amino acid sequence having 95% or more (e.g.,
96% or more, 97% or more, 98% or more, or 99% or more) sequence
identity to an amino acid sequence comprising SEQ ID NO: 17 and 18
linked by a peptide bond or a peptide linker sequence.
[0224] (76) The bispecific antibody of (75), wherein the
immunoglobulin Fc region comprises an amino acid sequence having
95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or
more) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.
[0225] (77) The bispecific antibody of any of (75) or (76), wherein
the PD-1 polypeptide variant is linked by a peptide linker sequence
to the N-terminus of the immunoglobulin Fc region.
[0226] (78) The bispecific antibody of any of (75) to (77), wherein
the scFv for the anti-4-1BB antibody is linked by peptide linker
sequence to the C-terminus of the immunoglobulin Fc region.
[0227] (79) The bispecific antibody of any of (75) to (78), wherein
the peptide linker sequence is selected from the group consisting
of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
[0228] (80) The bispecific antibody of any of (75) to (79), wherein
the scFv for the anti-4-1BB antibody comprises SEQ ID NO: 22 or SEQ
ID NO: 23 or a sequence that is at least 70% identical (e.g., at
least 75% identical, at least 80% identical, at least 85%
identical, at least 90% identical, at least 95% identical, at least
96% identical, at least 97% identical, at least 98% identical, at
least 99% identical, or 100% identical) to SEQ ID NO: 22 or SEQ ID
NO: 23.
[0229] (81) The bispecific antibody of any of (75) to (80), wherein
the bispecific antibody has increased binding affinity to a PD-L1
molecule as compared to a wild-type PD-1 polypeptide and specific
binding affinity to 4-1BB.
[0230] (82) A pharmaceutical composition comprising:
[0231] the bispecific antibody of any of (75) to (81); and
[0232] a pharmaceutically acceptable carrier.
[0233] (83) A method of treating a disease or a condition in a
subject in need thereof, the method comprising:
[0234] administering to the subject the pharmaceutical composition
of (82), thereby treating a disease or a condition.
[0235] (84) The method of (83), wherein the subject has cancer.
[0236] (85) The method of (84), wherein the cancer is selected from
a bladder cancer, breast cancer, cervical cancer, colon cancer,
endometrial cancer, esophageal cancer, fallopian tube cancer, gall
bladder cancer, gastrointestinal cancer, head and neck cancer,
hematological cancer, laryngeal cancer, liver cancer, lung cancer,
lymphoma, melanoma, mesothelioma, ovarian cancer, primary
peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer,
thyroid cancer, pancreatic cancer, renal cell carcinoma,
glioblastoma, and prostate cancer.
[0237] (86) The method of any of (83) to (85), wherein decreased or
no antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC) effects are observed.
[0238] (87) The method of any of (83) to (86), wherein decreased or
no hepatotoxicity is observed.
EXAMPLES
[0239] The disclosure is further described in the following
examples, which do not limit the scope of the disclosure described
in the claims.
Example 1--Variants of PD-1
[0240] PD-1 Fc fusion proteins including PD-1 polypeptide variants
were produced using a PD1-G.sub.4S linker-Fc_pcDNA3.3 plasmid. The
topology of human PD-1 protein is shown in Table 1. The sequences
of human PD-1 and PD-1 polypeptide variants are shown in FIG. 1 and
FIG. 2. Further, the mutations for each PD-1 polypeptide variant is
listed in Table 2, wherein the amino acid residue number is
relative to the full-length human PD-1 wild type sequence shown in
FIG. 1 (SEQ ID NO: 11). PD-1 polypeptide variants were designed to
include an extracellular domain (24-170) and a portion of a
transmembrane domain (171-172) of the full-length human PD-1 wild
type sequence shown in FIG. 1 (SEQ ID NO: 11). Specifically, the
PD-1 Fc fusion proteins were produced using an animal cell
expression vector pcDNA3.3 wherein restriction enzymes EcoRI and
BamHI were inserted at restriction enzyme sites. Human PD-1 signal
peptide (seq ID: Q15116, 1-23) was used for the signal peptide and
207aa-230aa IMGT allele IGHG1*03, 231aa-457aa IMGT allele IGHG2*01
was used for the Fc region. A DNA construct encoding a PD-1 Fc
fusion protein is shown in FIG. 3.
TABLE-US-00003 TABLE 1 Feature Key Position(s) Description
Topological domain 24-170 Extracellular Transmembrane 171-191
Helical Topological domain 192-288 Cytoplasmic
TABLE-US-00004 TABLE 2 Name Mutation Wild type PD-1 (SEQ ID NO: 11)
PD-1.m7 D26E, P34A, V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E,
N74G, K78T, Q99R, L122Y, A125V, A132I, R139G PD-1.m8 D26E, P34A,
V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E, N74G, K78T C93H,
Q99R, R114Q, L122Y, A125V, A132I, R139G euPD-1 D26E, P34A, V43L,
T45A, T59A, V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R,
R114Q, L122Y, A125V, A132I
Example 2--Analyzing and Characterizing PD-1 Polypeptide
Variants
[0241] PD-1 polypeptide variants were inserted into plasmids and
used to produce PD-1 Fc fusion proteins using Expi293 expression
system (Invitrogen). The polypeptide variants were then purified
using AktaPure (GE healthcare), and FibroPrismA column (GE
healthcare, Cat #17-0618-01). The purified polypeptide variants
were run through a desalting column (GE healthcare, Cat
#17-1408-01) and protein concentration was measured using Multiskan
GO (Thermo). Results are shown in Table 3.
TABLE-US-00005 TABLE 3 Concentration Volume Yield Ratio PD-1 type
(mg/mL) (mL) (mg) (wt/mt) PD-1 wild type 0.35 2 0.696 1 PD-1.m7
0.11 2 0.224 0.322 PD-1.m8 0.25 2 0.494 0.710 euPD-1 0.33 2 0.662
0.951
Example 3--Analyzing PD-1 Polypeptide Variants with SDS-PAGE
[0242] The PD-1 polypeptide variants were added to LDS sample
buffer (Invitrogen, Cat #B0007), wherein a sample reducing agent
(Invitrogen, Cat #B0004) was added to the reducing condition group
and incubated for 10 minutes at 70.degree. C. SDS running buffer
(Bio-rad, Cat #1610732) was added to the prepared samples and the
samples were run for 30 minutes using Mini-PROTEIN TGX Stain-Free
Gel (Bio-rad, Cat #456-8096). Results were analyzed using Chemidoc
(Bio-rad) (FIG. 4).
Example 4--Analyzing Affinity to PD-L1 Molecules
[0243] The affinity of PD-1 polypeptide variants to PD-L1 molecules
was analyzed using surface plasmon resonance (SPR). The PD-1
polypeptide variants were diluted to a concentration of 2 ug/mL
then fixed on CM5 chip (GE Healthcare, Cat #BR-1005-30). PD-L1
molecules (Sino, Cat #10084-H08H) were injected at concentrations
100, 50, 25, 12.5, 6.25, 3.125 nM with an association time of 150
seconds and dissociation time of 240 seconds. Biacore T200 (GE
healthcare) was used to measure and analyze affinity of each PD-1
polypeptide variant. Results show PD-1 polypeptide variant euPD-1
has highest binding affinity to PD-L1 (Table 4).
TABLE-US-00006 TABLE 4 Affinity to PD-L1 PD-1 type ka (1/Ms) kd
(1/s) K.sub.D (M) PD-1 2.373E+5 0.01018 4.289E-8 PD-1.m7 1.964E+6
0.002161 1.100E-9 PD-1.m8 1.757E+6 0.001822 1.037E-9 euPD-1
1.822E+6 0.001301 7.140E-10
Example 5--Size Exclusion Chromatography
[0244] The PD-1 polypeptide variants were analyzed using HPLC
(Agilent Technologies, 1260 infinity II LC system) and size
exclusion column (Tosoh, TSKgel G3000 SWXL, 7.8.times.300 mm, Part
No. 0008541, Column No. 004E04320E). Gel filtration standard
(BIO-RAD, Cat. #151-1901) was used for the control (FIGS.
5A-5E).
Example 6--PD-1 Fusion Proteins Cell Binding Assay
[0245] PD-L1 high expressed cell line MDA-MB-231 (human breast
cancer cell) and PD-L1 low expressed cell line MCF-7 (human breast
cancer cell) cells were used. FIG. 6 shows the expression level of
PD-L1 in each cell line. From FIG. 6 it is confirmed that PD-L1 is
highly expressed in MDA-MB-231 and not in MCF7.
[0246] 1.5.times.10.sup.5 cells were incubated at 4.degree. C. for
20 minutes with antibody treatment. The treatment concentrations of
euPD-1 Fc, PD-1 Fc, and Tecentriq (Genetech, atezolizumab,
anti-PD-L1 antibody) were serial dilution doubled from 877.19 nM to
a total of 12 points. After washing once with FACS washing buffer
(0.5% FBS+0.1% NaN.sub.3 in DPBS), anti-hFC-AF488 2nd antibody
(Jackson ImmunoResearch) was treated at 1 .mu.l/well for 20
minutes. After washing two additional times, FACS analysis was
performed.
[0247] As a result of FACS analysis, it was confirmed that there
was almost no background signal by the 2nd antibody. In FIG. 7A,
PD-1 Fc did not bind to PD-L1 positive cells in the experimental
concentration range, whereas euPD-1 Fc and Tecentriq were
dose-dependently bound to PD-L1 positive cells. As a result of FACS
analysis, in FIG. 7B, all three antibodies did not bind to PD-L1
negative cells.
Example 7--PD-1 Fusion Protein Antigen Binding Assay
[0248] Antigen binding assay was performed to confirm whether
euPD-1 Fc binds to antigen PD-L1 or PD-L2.
[0249] The assay method was performed as shown in FIGS. 8A and 8C.
Briefly, after coating PD-L1 antigen or PD-L2 antigen at a
concentration of 1 .mu.g/ml in a 96-well immunoplate at 4.degree.
C. overnight, 150 .mu.l of 1.times. assay buffer (Biolegend) was
treated for 1 hour to block non-specific binding. Each of euPD-1
Fc, PD-1 Fc, and Tecentriq was treated with 100 uL and incubated
for 2 hours. The treatment concentration on the antigen
PD-L1-coated plate was serially diluted from 30 .mu.g/mL to 3 times
to make a total of 15 points, The treatment concentration on the
antigen PD-L2 coated plate was serially diluted 3 times from 100
.mu.g/mL to a total of 12 points. Anti-hFc-HRP (Biolegend) was
treated with 100 .mu.l at a concentration of 0.4 .mu.g/mL. After
incubation for 1 hour, color was developed with TMB, and after 3
minutes, the reaction was stopped using sulfuric acid. Washing was
performed 3 times using washing buffer in all processes except for
the process after TMB treatment, All processes except for the
antigen coating process were carried out at room temperature. As a
result of the experiment, as shown in FIG. 8B, binding of euPD-1 Fc
and Tecentriq to antigen PD-L1 was confirmed in a dose-dependent
manner. And in FIG. 8D, only PD-1 Fc bound to antigen PD-L2 in a
dose-dependent manner, and euPD-1 Fc and Tecentriq did not
bind.
Example 8--PD-1 Fusion Protein Blockade Bioassay
[0250] A blockade bioassay was performed to verify the binding
inhibitory effect of PD-1 and PD-L1 (Bicytogen).
[0251] The bioassay product used (Promega, J1250) expresses
luciferase under conditions in which the binding of PD-1 and PD-L1
is inhibited. The bioassay and luciferase assay followed Promega
protocol. Target cells were seeded at 4.times.10.sup.4 cells/100
.mu.L/well in a 96 well white plate. After overnight incubation in
a 37.degree. C. CO.sub.2 incubator, antibodies and effector cells
were treated. As the antibody, euPD-1 Fc, PD-1 Fc, Tecentriq, and
Keytruda (Merck, pembrolizumab, anti-PD-1 antibody) were used. The
treatment concentration was serially dilution 2.5 times from 217.39
nM to make a total of 10 points. After 6 hours of incubation in a
37.degree. C. CO.sub.2 incubator, luciferin was treated and
luciferase assay was performed. As a result of luciferase assay, as
shown in FIG. 9A, euPD-1 Fc, Tecentriq, and Keytruda showed
inhibitory effects in a dose-dependent manner, and, as shown in
FIG. 9B, euPD-1 Fc showed more than 2.4 to 4.9 times more
inhibitory effect than PD-1 Fc.
Example 9--PD-1 Fusion Protein In Vivo Efficacy Study
[0252] To verify the efficacy of euPD-1 Fc, a tumor growth
inhibition in vivo study was performed. The experiment was
performed using a female C57BL/6 mouse that knocked-in hPD-1.
[0253] As the tumor cells, MC38 cells expressing human PD-L1 were
used as shown in FIG. 10, and 8.times.10.sup.6 tumor cells per
individual were subcutaneously administered. After one week of
administration, the tumor size was measured and the administration
groups were allocated so that the mean and standard deviation were
similar (i.e., about 100 mm.sup.3). Antibodies euPD-1 Fc, PD-1 Fc,
and Tecentriq three were injected by i.v. As a negative control,
the excipient DPBS (Gibco) was used. The antibody dose was set to 5
mg/kg and 2 mg/kg based on euPD-1 Fc, and 8.77 .mu.M concentration
was used for PD-1 fc and tecentriq 5 mg/kg in consideration of
molecular weight. The administration was carried out at 100 .mu.l,
and was administered 5 times at 3-day intervals. The tumor size was
also measured at 3-day or 4-day intervals, and the tumor size was
measured until 2 days after the last administration. In addition,
blood was collected on the day of the end of the experiment to
confirm toxicity, and the concentrations of ALT (alanine
aminotransferase), AST (aspartate aminotransferase), BUN (blood
urea nitrogen), and T-BIL (total Bilirubin) were checked using a
biochemical analyzer.
[0254] As a result of tumor size observation, as shown in FIG. 11A
and FIG. 11B, both euPD-1 Fc (8.77 .mu.M) and tecentriq (8.77
.mu.M) inhibited tumor growth compared to administration of
negative control; PD-1 Fc (8.77 .mu.M) showed growth similar to
that of the negative control. In addition, as shown in FIG. 11C and
FIG. 11D, euPD-1 Fc reduced the tumor size by 33.9% compared to the
negative control administration in the low concentration (2 mg/kg)
administration group, A 66.1% tumor size reduction was observed in
the group administered with a high grade (5 mg/kg), showing a
dose-dependent inhibitory effect.
[0255] Further, a liver toxicity index analysis was performed and
shown in FIG. 12. As shown in FIG. 12 for the liver toxicity index
analysis, in the euPD-1 Fc administration group, all four
indicators were in the normal range (ALT: 17-77 U/L (FIG. 12A and
FIG. 12E), AST: 54-298 U/L (FIG. 12B and FIG. 12F), BUN: 8-33 mg/dL
(FIG. 12C and FIG. 12G), T-BIL: 8-33 mg/dL (FIG. 12D and FIG.
12H)), no hepatotoxicity was confirmed within the experimental
conditions.
[0256] Thus, it was confirmed that the fusion proteins and
bispecific antibodies of the present invention comprising euPD-1
and IgG1 variants displayed no hepatotoxicity. In addition, the
antibodies of the present invention lack ADCC and CDC effects.
Example 10--Design, Production, and Characterization of PD-1 Fusion
Protein euPD-1 BsAb
[0257] Fc fusion BsAb using euPD-1 and anti-4-1BB antibody was
designed as shown in FIG. 13. A (G.sub.4S).sub.2 linker was used to
link euPD-1 and Fc, and Fc region modified with IgG1 was used for
the Fc region used for BsAb. (L234A, L235A, K322A, D356E,
L358M).
[0258] BsAb in the form of linking two euPD-1s was also designed;
to link two euPD-1s, a (G.sub.4S).sub.3 linker was used and named
euPD-1.2.
[0259] On the C-terminal side, the anti-4-1BB antibody was bound to
Fc in the form of scFv; at this time, it was linked with a
(G.sub.4S).sub.2 linker, and two types of scFvs for the anti-4-1BB
antibody, VH-218 linker-VL (HLC 218) and VL-218 linker-VH (LHC
218), were used.
[0260] The constructs in Table 5 or FIG. 13 were produced. Also
provided are the sequences referred to above.
TABLE-US-00007 TABLE 5 Seq ID No. Name Amino acid sequence 12
euPD-1 x FLESPDRPWNAPTFSPALLLVAEGDNATFTCSFSNASESFHVV 94kvt HLC
WHRESPSGQTDTLAAFPEDRSQPGQDHRFRVTRLPNGRDFH 218
MSVVRAQRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRA
EVPTAHPSPSPRPAGQFQTLVVGASGGGGSGGGGSDKTHTCP
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGAS
VKLSCKASGYTFSSYWMHWVRQAPGQGLEWIGEINPGNGH
TNYNEKFKSRVTMTRDTSTSTAYMELSSLRSEDTAVYYCAR
SFKTARAFAYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGDIV
MTQSPAFLSVTPGEKVTITCRASQTISDYLHWYQQKPDQAPK
LLIKYASQSISGIPSRFSGSGSGTDFTFTISSLEAEDAATYYCQ DGHSWPPTFGQGTKLEIK 13
euPD-1.2 x FLESPDRPWNAPTFSPALLLVAEGDNATFTCSFSNASESFHVV 94kvt HLC
WHRESPSGQTDTLAAFPEDRSQPGQDHRFRVTRLPNGRDFH 218
MSVVRAQRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRA
EVPTAHPSPSPRPAGQFQTLVVGGGGGSGGGGSGGGGSFLES
PDRPWNAPTFSPALLLVAEGDNATFTCSFSNASESFHVVWHR
ESPSGQTDTLAAFPEDRSQPGQDHRFRVTRLPNGRDFHMSV
VRAQRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRAEVPT
AHPSPSPRPAGQFQTLVVGASGGGGSGGGGSDKTHTCPPCPA
PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY
TQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGASVKLS
CKASGYTFSSYWMHWVRQAPGQGLEWIGEINPGNGHTNYN
EKFKSRVTMTRDTSTSTAYMELSSLRSEDTAVYYCARSFKTA
RAFAYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGDIVMTQS
PAFLSVTPGEKVTITCRASQTISDYLHWYQQKPDQAPKLLIK
YASQSISGIPSRFSGSGSGTDFTFTISSLEAEDAATYYCQDGHS WPPTFGQGTKLEIK 14
euPD-1 x FLESPDRPWNAPTFSPALLLVAEGDNATFTCSFSNASESFHVV 94kvt LHC
WHRESPSGQTDTLAAFPEDRSQPGQDHRFRVTRLPNGRDFH 218
MSVVRAQRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRA
EVPTAHPSPSPRPAGQFQTLVVGASGGGGSGGGGSDKTHTCP
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGGGGGSGGGGSDIVMTQSPAFLSVTPGEK
VTITCRASQTISDYLHWYQQKPDQAPKLLIKYASQSISGIPSRF
SGSGSGTDFTFTISSLEAEDAATYYCQDGHSWPPTFGQGTKL
EIKGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKLS
CKASGYTFSSYWMHWVRQAPGQGLEWIGEINPGNGHTNYN
EKFKSRVTMTRDTSTSTAYMELSSLRSEDTAVYYCARSFKTA RAFAYWGQGTLVTVSS 15
euPD-1.2 x FLESPDRPWNAPTFSPALLLVAEGDNATFTCSFSNASESFHVV 94kvt LHC
WHRESPSGQTDTLAAFPEDRSQPGQDHRFRVTRLPNGRDFH 218
MSVVRAQRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRA
EVPTAHPSPSPRPAGQFQTLVVGGGGGSGGGGSGGGGSFLES
PDRPWNAPTFSPALLLVAEGDNATFTCSFSNASESFHVVWHR
ESPSGQTDTLAAFPEDRSQPGQDHRFRVTRLPNGRDFHMSV
VRAQRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRAEVPT
AHPSPSPRPAGQFQTLVVGASGGGGSGGGGSDKTHTCPPCPA
PEAAGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY
TQKSLSLSPGGGGGSGGGGSDIVMTQSPAFLSVTPGEKVTITC
RASQTISDYLHWYQQKPDQAPKLLIKYASQSISGIPSRFSGSG
SGTDFTFTISSLEAEDAATYYCQDGHSWPPTFGQGTKLEIKGS
TSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKLSCKAS
GYTFSSYWMHWVRQAPGQGLEWIGEINPGNGHTNYNEKFK
SRVTMTRDTSTSTAYMELSSLRSEDTAVYYCARSFKTARAF AYWGQGTLVTVSS 16 Fc
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 17
94kvt VH QVQLVQSGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQ
APGQGLEWIGEINPGNGHTNYNEKFKSRVTMTRDTSTSTAY
MELSSLRSEDTAVYYCARSFKTARAFAYWGQGTLVTVSS 18 94kvt VL
DIVMTQSPAFLSVTPGEKVTITCRASQTISDYLHWYQQKPDQ
APKLLIKYASQSISGIPSRFSGSGSGTDFTFTISSLEAEDAATYY CQDGHSWPPTFGQGTKLEIK
19 (G.sub.4S).sub.2 GGGGSGGGGS 20 (G.sub.4S).sub.3 GGGGSGGGGSGGGGS
21 218 GSTSGSGKPGSGEGSTKG 22 94kvt HLC
QVQLVQSGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQ 218
APGQGLEWIGEINPGNGHTNYNEKFKSRVTMTRDTSTSTAY
MELSSLRSEDTAVYYCARSFKTARAFAYWGQGTLVTVSSGS
TSGSGKPGSGEGSTKGDIVMTQSPAFLSVTPGEKVTITCRASQ
TISDYLHWYQQKPDQAPKLLIKYASQSISGIPSRFSGSGSGTD
FTFTISSLEAEDAATYYCQDGHSWPPTFGQGTKLEIK 23 94kvt LHC
DIVMTQSPAFLSVTPGEKVTITCRASQTISDYLHWYQQKPDQ 218
APKLLIKYASQSISGIPSRFSGSGSGTDFTFTISSLEAEDAATYY
CQDGHSWPPTFGQGTKLEIKGSTSGSGKPGSGEGSTKGQVQL
VQSGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQG
LEWIGEINPGNGHTNYNEKFKSRVTMTRDTSTSTAYMELSSL
RSEDTAVYYCARSFKTARAFAYWGQGTLVTVSS
[0261] Transient transfection was performed into Expi293F cells to
produce euPD-1 BsAbs (bispecific antibodies); purification was
carried out by protein A column.
[0262] As a result of SDS-PAGE analysis, in the non-reducing
condition, euPD-1.times.94kvt HLC 218 and euPD-1.times.94kvt LHC
218 with one euPD-1 were observed to be about 160 kDa; in the
reducing condition, it was observed to be about 80 kDa. The euPD-1
doubled form, euPD-1.2.times.94 kvt HLC 218 and euPD-1.2.times.94
kvt LHC 218, is observed to be about 250 kDa; in the reducing
condition, it was observed to be about 125 kDa. As a result of size
exclusion chromatography analysis, a single peak with a purity of
90% or more was observed (see FIG. 15 where FIG. 15A is a standard
curve, FIG. 15B is euPD-1.times.94kvt HLC 218, FIG. 15C is
euPD-1.times.94kvt LHC 218, FIG. 15D is euPD-1.2.times.94kvt HLC
218, and FIG. 15E is euPD-1.2.times.94kvt LHC 218).
[0263] Surface plasmon resonance (SPR) analysis similar to as
described in Example 4 was followed. Specifically, 25 .mu.g/ml
anti-human Fc antibody was immobilized on the CM5 chip. 25 .mu.g/ml
anti-human Fc antibody was immobilized on the CM5 chip. After that,
4 types of euPD-1 BsAb were caused to flow at a flow rate of 10
.mu.g/ml and 10 .mu.g/min for 60 seconds to capture, and KD
analysis was performed using 100, 50, 25, 12.5, 3.25, 3.125, 0 nM
of PD-L1 antigen. FIG. 16 shows the SPR result of the euPD-1 BsAb
constructs.
Example 11--Antigen Binding Assay with euPD-1 BsAb Constructs
[0264] To check whether euPD-1 BsAbs binds to antigen PD-L1, an
antigen binding assay was performed.
[0265] The assay method was performed as shown in FIG. 17A.
Briefly, after coating PD-L1 antigen at a concentration of 1
.mu.g/ml in a 96-well immunoplate at 4.degree. C. with overnight,
150 .mu.l of 1.times. assay buffer (Biolegend) was treated for 1
hour to block non-specific binding. euPD-1 BsAb was incubated for 2
hours after treatment with 100 .mu.L. The treatment concentration
was serially diluted 10 times from 10 .mu.g/mL to a total of 3
points. 100 .mu.l of biontinylated 4-1BB at a concentration of 1
.mu.g/ml was treated for 1 hour. Avidin-HRP (Bioglend) was treated
with 100 .mu.l at the concentration shown in the protocol for 30
minutes. After color development with TMB, the reaction was stopped
with sulfuric acid after 3 minutes. Washing was performed 3 times
using washing buffer in all processes except for the process after
TMB treatment; except for the PD-L1 coating process, all processes
were carried out at room temperature.
[0266] As a result of the experiment, as shown FIG. 17B, binding of
euPD-1 BsAb to antigen PD-L1 was confirmed in a dose-dependent
manner.
Example 12--4-1BB/PD-1 Combination Bioassay
[0267] In order to verify the effect of inhibiting the binding of
PD-1 and PD-L1 and the effect of 4-1BB activity, a 4-1BB/PD-1
combination bioassay was performed.
[0268] For this experiment, the bioassay product (Promega,
CS1978I10) is an assay system which expresses luciferase when 4-1BB
is activated by 4-1BB antibody stimulation and when the PD-1/PD-L1
interaction inhibits at the same time. The bioassay and luciferase
assay followed Promega protocol. MDA-MB-231 cells expressing PD-L1
were seeded at 4.times.10.sup.4 cells/100 .mu.L/well in a 96 well
white plate. After 0/N incubation in a 37.degree. C. CO.sub.2
incubator, antibodies and PD1+4-1BB effector cells were treated. As
antibodies, euPD-1.times.94 kvt HLC 218 and euPD-1.times.94 kvt LHC
218 were used. The treatment concentration was serially dilution
from 60 ng/ml to 4 times to make a total of 4 points. After 6 hours
of incubation in a 37.degree. C. CO.sub.2 incubator, luciferin was
treated and luciferase assay was performed.
[0269] As a result of luciferase assay, as shown in FIG. 18,
euPD-1.times.94 kvt HLC 218 and euPD-1.times.94 kvt LHC 218
activated 4-1BB while inhibiting PD-1/PD-L1 in a dose-dependent
manner.
Sequence CWU 1
1
231447DNAHomo sapiens 1tttttggatt ctccagaccg gccttggaac ccgcccacgt
ttagccctgc tcttttggta 60gttacagagg gggacaacgc cacattcacc tgcagcttct
ctaatacgtc cgagagcttt 120gtactgaatt ggtatagaat gagtccatct
aatcagacag ataaattggc tgccttccct 180gaagacagga gtcagccggg
tcaggactgc agattccgcg ttacgcaact cccaaatggt 240cgagactttc
atatgtcagt tgttcgggcg aggagaaatg atagcggtac ttacctgtgc
300ggcgcgatat ctctcgcacc aaaagcacag attaaagagt ctctccgggc
tgaactccgc 360gtgacagaaa ggcgagccga ggtaccaacg gcgcacccat
caccgagtcc tagacctgcg 420ggccaattcc agactttggt tgtcgga
4472149PRTHomo sapiens 2Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro
Pro Thr Phe Ser Pro1 5 10 15Ala Leu Leu Val Val Thr Glu Gly Asp Asn
Ala Thr Phe Thr Cys Ser 20 25 30Phe Ser Asn Thr Ser Glu Ser Phe Val
Leu Asn Trp Tyr Arg Met Ser 35 40 45Pro Ser Asn Gln Thr Asp Lys Leu
Ala Ala Phe Pro Glu Asp Arg Ser 50 55 60Gln Pro Gly Gln Asp Cys Arg
Phe Arg Val Thr Gln Leu Pro Asn Gly65 70 75 80Arg Asp Phe His Met
Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly 85 90 95Thr Tyr Leu Cys
Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys 100 105 110Glu Ser
Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val 115 120
125Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln
130 135 140Thr Leu Val Val Gly1453447DNAArtificial SequencePD-1
polypeptide variant PD-1.m7 3tttctggagt cacccgaccg cccttggaac
gcacctacct tttccccggc cctcttgctg 60gtcgcagaag gagataacgc cactttcaca
tgcagcttta gtaacgcctc cgaatctttt 120catgtagttt ggcacagaga
aagcccctcc ggacaaaccg acaccttggc tgcgtttccc 180gaagaccgaa
gtcaaccagg gcaggactgc cggttccgcg taacacggct gccaaacggt
240agggacttcc acatgtcagt ggttcgagca cgccggaacg acagcgggac
gtatgtctgc 300ggagtcatta gccttgcccc gaagatacag attaaagaaa
gtcttggggc agaacttcga 360gtcaccgagc gcagggccga ggtcccaacg
gcacatccca gtcctagtcc acggcccgcc 420ggtcaatttc agacccttgt agtgggc
4474149PRTArtificial SequencePD-1 polypeptide variant PD-1.m7 4Phe
Leu Glu Ser Pro Asp Arg Pro Trp Asn Ala Pro Thr Phe Ser Pro1 5 10
15Ala Leu Leu Leu Val Ala Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser
20 25 30Phe Ser Asn Ala Ser Glu Ser Phe His Val Val Trp His Arg Glu
Ser 35 40 45Pro Ser Gly Gln Thr Asp Thr Leu Ala Ala Phe Pro Glu Asp
Arg Ser 50 55 60Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Arg Leu
Pro Asn Gly65 70 75 80Arg Asp Phe His Met Ser Val Val Arg Ala Arg
Arg Asn Asp Ser Gly 85 90 95Thr Tyr Val Cys Gly Val Ile Ser Leu Ala
Pro Lys Ile Gln Ile Lys 100 105 110Glu Ser Leu Gly Ala Glu Leu Arg
Val Thr Glu Arg Arg Ala Glu Val 115 120 125Pro Thr Ala His Pro Ser
Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln 130 135 140Thr Leu Val Val
Gly1455447DNAArtificial SequencePD-1 polypeptide variant PD-1.m8
5tttttggaaa gtcccgatcg gccttggaac gctcccacat ttagcccggc cctgcttttg
60gttgctgaag gcgataacgc cacttttaca tgcagtttca gcaacgcctc tgaaagtttc
120catgtagtgt ggcaccgcga gtctccaagt gggcaaacag atacccttgc
agctttcccg 180gaagatagga gtcagccagg gcaggatcac cggtttagag
tcactcgcct ccccaatggt 240agagattttc acatgagcgt cgttcgagct
cagagaaacg atagtggcac atacgtttgt 300ggcgtaatat ctctcgcccc
gaagatccag attaaagagt cccttggcgc ggagctgaga 360gtcaccgaga
ggcgagctga ggtgcctaca gctcatccta gcccgagccc aaggccagct
420ggacagttcc aaactttggt tgtaggc 4476149PRTArtificial SequencePD-1
polypeptide variant PD-1.m8 6Phe Leu Glu Ser Pro Asp Arg Pro Trp
Asn Ala Pro Thr Phe Ser Pro1 5 10 15Ala Leu Leu Leu Val Ala Glu Gly
Asp Asn Ala Thr Phe Thr Cys Ser 20 25 30Phe Ser Asn Ala Ser Glu Ser
Phe His Val Val Trp His Arg Glu Ser 35 40 45Pro Ser Gly Gln Thr Asp
Thr Leu Ala Ala Phe Pro Glu Asp Arg Ser 50 55 60Gln Pro Gly Gln Asp
His Arg Phe Arg Val Thr Arg Leu Pro Asn Gly65 70 75 80Arg Asp Phe
His Met Ser Val Val Arg Ala Gln Arg Asn Asp Ser Gly 85 90 95Thr Tyr
Val Cys Gly Val Ile Ser Leu Ala Pro Lys Ile Gln Ile Lys 100 105
110Glu Ser Leu Gly Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val
115 120 125Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln
Phe Gln 130 135 140Thr Leu Val Val Gly1457447DNAArtificial
SequencePD-1 polypeptide variant euPD-1 7tttctcgaat caccggacag
accctggaat gcgcccacat tctcaccagc acttttgctg 60gtagcagagg gcgataatgc
tacattcacg tgttccttca gtaatgcaag cgagtcattt 120catgtggttt
ggcatcgaga gtcacctagt gggcagactg atacacttgc cgcattcccg
180gaagatcgct cccagccagg tcaggatcac cggttcaggg taacccgact
gccgaatggg 240cgcgatttcc atatgagcgt tgtccgggcg caacggaacg
atagtggaac atacgtgtgt 300ggcgtaatat ccctcgctcc caaaatacaa
ataaaggagt ctctgagagc agagctgaga 360gtgacagaac gacgggcgga
agttcccacg gctcatccgt caccaagtcc gcgccccgca 420ggccaatttc
aaacgctcgt cgtaggc 4478149PRTArtificial SequencePD-1 polypeptide
variant euPD-1 8Phe Leu Glu Ser Pro Asp Arg Pro Trp Asn Ala Pro Thr
Phe Ser Pro1 5 10 15Ala Leu Leu Leu Val Ala Glu Gly Asp Asn Ala Thr
Phe Thr Cys Ser 20 25 30Phe Ser Asn Ala Ser Glu Ser Phe His Val Val
Trp His Arg Glu Ser 35 40 45Pro Ser Gly Gln Thr Asp Thr Leu Ala Ala
Phe Pro Glu Asp Arg Ser 50 55 60Gln Pro Gly Gln Asp His Arg Phe Arg
Val Thr Arg Leu Pro Asn Gly65 70 75 80Arg Asp Phe His Met Ser Val
Val Arg Ala Gln Arg Asn Asp Ser Gly 85 90 95Thr Tyr Val Cys Gly Val
Ile Ser Leu Ala Pro Lys Ile Gln Ile Lys 100 105 110Glu Ser Leu Arg
Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val 115 120 125Pro Thr
Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln 130 135
140Thr Leu Val Val Gly1459720DNAArtificial SequenceFc (207-230 IMGT
allele IGHG1*03, 231- 457 IMGT allele IGHG2*01) 9agcaacacta
aagtcgacaa gcgagtagaa ccgaaatcat gcgacaaaac acatacgtgc 60cctccctgcc
cagcaccacc tgtcgcgggc ccctctgttt tcctgtttcc acccaagcca
120aaggacacat tgatgatttc ccggactcct gaagtcacct gcgtggtagt
agatgtatca 180catgaagatc cagaagtcca gttcaactgg tatgtggacg
gagtagaggt acataatgcc 240aagaccaaac cacgggaaga gcagttcaac
agtactttcc gggtagttag cgttttgact 300gtcgtacacc aagactggct
taatggaaaa gaatacaagt gtaaggtaag caacaagggc 360ctgccggctc
cgatagagaa aaccattagc aagacaaagg gccaaccacg cgaaccccag
420gtatataccc tcccaccgtc ccgcgaggag atgactaaga atcaagtttc
tctcacgtgc 480ttggtaaagg gcttctatcc gagcgatata gccgtggagt
gggagtctaa tggtcagccc 540gaaaacaatt acaaaactac gcctcctatg
ctggacagtg atgggagctt ctttctttac 600agtaagctta ccgtggacaa
gtctcggtgg caacaaggaa atgtttttag ttgttctgta 660atgcatgaag
cacttcataa ccattacacc cagaaaagtc tgagcttgtc cccgggaaaa
72010240PRTArtificial SequenceFc (207-230 IMGT allele IGHG1*03,
231- 457 IMGT allele IGHG2*01) 10Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys1 5 10 15Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Pro Val Ala Gly Pro Ser 20 25 30Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 35 40 45Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 50 55 60Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala65 70 75 80Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val 85 90 95Ser
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 100 105
110Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
115 120 125Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 130 135 140Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys145 150 155 160Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 165 170 175Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Met Leu Asp 180 185 190Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 195 200 205Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 210 215 220Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys225 230
235 24011288PRTHomo Sapience 11Met Gln Ile Pro Gln Ala Pro Trp Pro
Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg Pro Gly Trp Phe
Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro Thr Phe Ser Pro
Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala Thr Phe Thr Cys
Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu Asn Trp Tyr Arg
Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75 80Ala Phe Pro
Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95Val Thr
Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105
110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu
Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro
Ser Pro Ser Pro145 150 155 160Arg Pro Ala Gly Gln Phe Gln Thr Leu
Val Val Gly Val Val Gly Gly 165 170 175Leu Leu Gly Ser Leu Val Leu
Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190Ser Arg Ala Ala Arg
Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205Leu Lys Glu
Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220Glu
Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro225 230
235 240Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser
Gly 245 250 255Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp
Gly Pro Arg 260 265 270Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His
Cys Ser Trp Pro Leu 275 280 28512641PRTArtificial SequenceeuPD-1 x
94kvt HLC 218 12Phe Leu Glu Ser Pro Asp Arg Pro Trp Asn Ala Pro Thr
Phe Ser Pro1 5 10 15Ala Leu Leu Leu Val Ala Glu Gly Asp Asn Ala Thr
Phe Thr Cys Ser 20 25 30Phe Ser Asn Ala Ser Glu Ser Phe His Val Val
Trp His Arg Glu Ser 35 40 45Pro Ser Gly Gln Thr Asp Thr Leu Ala Ala
Phe Pro Glu Asp Arg Ser 50 55 60Gln Pro Gly Gln Asp His Arg Phe Arg
Val Thr Arg Leu Pro Asn Gly65 70 75 80Arg Asp Phe His Met Ser Val
Val Arg Ala Gln Arg Asn Asp Ser Gly 85 90 95Thr Tyr Val Cys Gly Val
Ile Ser Leu Ala Pro Lys Ile Gln Ile Lys 100 105 110Glu Ser Leu Arg
Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val 115 120 125Pro Thr
Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln 130 135
140Thr Leu Val Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly145 150 155 160Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala 165 170 175Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 180 185 190Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 195 200 205His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 210 215 220Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr225 230 235 240Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 245 250
255Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro
260 265 270Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 275 280 285Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 290 295 300Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val305 310 315 320Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro 325 330 335Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 340 345 350Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 355 360 365Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 370 375
380Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
Gln385 390 395 400Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala Ser Val Lys 405 410 415Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Ser Ser Tyr Trp Met His 420 425 430Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Ile Gly Glu Ile 435 440 445Asn Pro Gly Asn Gly His
Thr Asn Tyr Asn Glu Lys Phe Lys Ser Arg 450 455 460Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu465 470 475 480Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser 485 490
495Phe Lys Thr Ala Arg Ala Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
500 505 510Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly
Ser Gly 515 520 525Glu Gly Ser Thr Lys Gly Asp Ile Val Met Thr Gln
Ser Pro Ala Phe 530 535 540Leu Ser Val Thr Pro Gly Glu Lys Val Thr
Ile Thr Cys Arg Ala Ser545 550 555 560Gln Thr Ile Ser Asp Tyr Leu
His Trp Tyr Gln Gln Lys Pro Asp Gln 565 570 575Ala Pro Lys Leu Leu
Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile 580 585 590Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr 595 600 605Ile
Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asp 610 615
620Gly His Ser Trp Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile625 630 635 640Lys13805PRTArtificial SequenceeuPD-1.2 x 94kvt
HLC 218 13Phe Leu Glu Ser Pro Asp Arg Pro Trp Asn Ala Pro Thr Phe
Ser Pro1 5 10 15Ala Leu Leu Leu Val Ala Glu Gly Asp Asn Ala Thr Phe
Thr Cys Ser 20 25 30Phe Ser Asn Ala Ser Glu Ser Phe His Val Val Trp
His Arg Glu Ser 35 40 45Pro Ser Gly Gln Thr Asp Thr Leu Ala Ala Phe
Pro Glu Asp Arg Ser 50 55 60Gln Pro Gly Gln Asp His Arg Phe Arg Val
Thr Arg Leu Pro Asn Gly65 70 75 80Arg Asp Phe His Met Ser Val Val
Arg Ala Gln Arg Asn Asp Ser Gly 85 90 95Thr Tyr Val Cys Gly Val Ile
Ser Leu Ala Pro Lys Ile Gln Ile Lys 100 105 110Glu Ser Leu Arg Ala
Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val 115 120 125Pro Thr Ala
His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln 130 135 140Thr
Leu Val Val Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly145 150
155 160Gly Gly Gly Ser Phe Leu Glu Ser Pro Asp Arg Pro Trp Asn Ala
Pro 165 170 175Thr Phe Ser Pro Ala Leu Leu Leu Val Ala Glu Gly Asp
Asn Ala Thr 180 185 190Phe
Thr Cys Ser Phe Ser Asn Ala Ser Glu Ser Phe His Val Val Trp 195 200
205His Arg Glu Ser Pro Ser Gly Gln Thr Asp Thr Leu Ala Ala Phe Pro
210 215 220Glu Asp Arg Ser Gln Pro Gly Gln Asp His Arg Phe Arg Val
Thr Arg225 230 235 240Leu Pro Asn Gly Arg Asp Phe His Met Ser Val
Val Arg Ala Gln Arg 245 250 255Asn Asp Ser Gly Thr Tyr Val Cys Gly
Val Ile Ser Leu Ala Pro Lys 260 265 270Ile Gln Ile Lys Glu Ser Leu
Arg Ala Glu Leu Arg Val Thr Glu Arg 275 280 285Arg Ala Glu Val Pro
Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala 290 295 300Gly Gln Phe
Gln Thr Leu Val Val Gly Ala Ser Gly Gly Gly Gly Ser305 310 315
320Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
325 330 335Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro 340 345 350Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val 355 360 365Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val 370 375 380Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln385 390 395 400Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln 405 410 415Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 420 425 430Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 435 440
445Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
450 455 460Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser465 470 475 480Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr 485 490 495Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr 500 505 510Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe 515 520 525Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 530 535 540Ser Leu Ser
Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly545 550 555
560Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
565 570 575Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Ser Ser 580 585 590Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp 595 600 605Ile Gly Glu Ile Asn Pro Gly Asn Gly His
Thr Asn Tyr Asn Glu Lys 610 615 620Phe Lys Ser Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Ala625 630 635 640Tyr Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 645 650 655Cys Ala Arg
Ser Phe Lys Thr Ala Arg Ala Phe Ala Tyr Trp Gly Gln 660 665 670Gly
Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys 675 680
685Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Val Met Thr Gln
690 695 700Ser Pro Ala Phe Leu Ser Val Thr Pro Gly Glu Lys Val Thr
Ile Thr705 710 715 720Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr Leu
His Trp Tyr Gln Gln 725 730 735Lys Pro Asp Gln Ala Pro Lys Leu Leu
Ile Lys Tyr Ala Ser Gln Ser 740 745 750Ile Ser Gly Ile Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp 755 760 765Phe Thr Phe Thr Ile
Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr 770 775 780Tyr Cys Gln
Asp Gly His Ser Trp Pro Pro Thr Phe Gly Gln Gly Thr785 790 795
800Lys Leu Glu Ile Lys 80514641PRTArtificial SequenceeuPD-1 x 94kvt
LHC 218 14Phe Leu Glu Ser Pro Asp Arg Pro Trp Asn Ala Pro Thr Phe
Ser Pro1 5 10 15Ala Leu Leu Leu Val Ala Glu Gly Asp Asn Ala Thr Phe
Thr Cys Ser 20 25 30Phe Ser Asn Ala Ser Glu Ser Phe His Val Val Trp
His Arg Glu Ser 35 40 45Pro Ser Gly Gln Thr Asp Thr Leu Ala Ala Phe
Pro Glu Asp Arg Ser 50 55 60Gln Pro Gly Gln Asp His Arg Phe Arg Val
Thr Arg Leu Pro Asn Gly65 70 75 80Arg Asp Phe His Met Ser Val Val
Arg Ala Gln Arg Asn Asp Ser Gly 85 90 95Thr Tyr Val Cys Gly Val Ile
Ser Leu Ala Pro Lys Ile Gln Ile Lys 100 105 110Glu Ser Leu Arg Ala
Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val 115 120 125Pro Thr Ala
His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln 130 135 140Thr
Leu Val Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly145 150
155 160Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala 165 170 175Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu 180 185 190Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser 195 200 205His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu 210 215 220Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr225 230 235 240Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 245 250 255Gly Lys
Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro 260 265
270Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
275 280 285Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val 290 295 300Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val305 310 315 320Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 325 330 335Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 340 345 350Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 355 360 365Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 370 375 380Ser
Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val385 390
395 400Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly Glu Lys
Val 405 410 415Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr
Leu His Trp 420 425 430Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Leu
Leu Ile Lys Tyr Ala 435 440 445Ser Gln Ser Ile Ser Gly Ile Pro Ser
Arg Phe Ser Gly Ser Gly Ser 450 455 460Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Leu Glu Ala Glu Asp Ala465 470 475 480Ala Thr Tyr Tyr
Cys Gln Asp Gly His Ser Trp Pro Pro Thr Phe Gly 485 490 495Gln Gly
Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys 500 505
510Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln
515 520 525Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Leu
Ser Cys 530 535 540Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met
His Trp Val Arg545 550 555 560Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile Gly Glu Ile Asn Pro Gly 565 570 575Asn Gly His Thr Asn Tyr Asn
Glu Lys Phe Lys Ser Arg Val Thr Met 580 585 590Thr Arg Asp Thr Ser
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu 595 600 605Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Phe Lys Thr 610 615 620Ala
Arg Ala Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser625 630
635 640Ser15805PRTArtificial SequenceeuPD-1.2 x 94kvt LHC 218 15Phe
Leu Glu Ser Pro Asp Arg Pro Trp Asn Ala Pro Thr Phe Ser Pro1 5 10
15Ala Leu Leu Leu Val Ala Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser
20 25 30Phe Ser Asn Ala Ser Glu Ser Phe His Val Val Trp His Arg Glu
Ser 35 40 45Pro Ser Gly Gln Thr Asp Thr Leu Ala Ala Phe Pro Glu Asp
Arg Ser 50 55 60Gln Pro Gly Gln Asp His Arg Phe Arg Val Thr Arg Leu
Pro Asn Gly65 70 75 80Arg Asp Phe His Met Ser Val Val Arg Ala Gln
Arg Asn Asp Ser Gly 85 90 95Thr Tyr Val Cys Gly Val Ile Ser Leu Ala
Pro Lys Ile Gln Ile Lys 100 105 110Glu Ser Leu Arg Ala Glu Leu Arg
Val Thr Glu Arg Arg Ala Glu Val 115 120 125Pro Thr Ala His Pro Ser
Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln 130 135 140Thr Leu Val Val
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly145 150 155 160Gly
Gly Gly Ser Phe Leu Glu Ser Pro Asp Arg Pro Trp Asn Ala Pro 165 170
175Thr Phe Ser Pro Ala Leu Leu Leu Val Ala Glu Gly Asp Asn Ala Thr
180 185 190Phe Thr Cys Ser Phe Ser Asn Ala Ser Glu Ser Phe His Val
Val Trp 195 200 205His Arg Glu Ser Pro Ser Gly Gln Thr Asp Thr Leu
Ala Ala Phe Pro 210 215 220Glu Asp Arg Ser Gln Pro Gly Gln Asp His
Arg Phe Arg Val Thr Arg225 230 235 240Leu Pro Asn Gly Arg Asp Phe
His Met Ser Val Val Arg Ala Gln Arg 245 250 255Asn Asp Ser Gly Thr
Tyr Val Cys Gly Val Ile Ser Leu Ala Pro Lys 260 265 270Ile Gln Ile
Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg 275 280 285Arg
Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala 290 295
300Gly Gln Phe Gln Thr Leu Val Val Gly Ala Ser Gly Gly Gly Gly
Ser305 310 315 320Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala 325 330 335Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro 340 345 350Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val 355 360 365Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 370 375 380Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln385 390 395 400Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 405 410
415Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala
420 425 430Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro 435 440 445Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr 450 455 460Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser465 470 475 480Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr 485 490 495Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 500 505 510Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 515 520 525Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 530 535
540Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly
Gly545 550 555 560Ser Asp Ile Val Met Thr Gln Ser Pro Ala Phe Leu
Ser Val Thr Pro 565 570 575Gly Glu Lys Val Thr Ile Thr Cys Arg Ala
Ser Gln Thr Ile Ser Asp 580 585 590Tyr Leu His Trp Tyr Gln Gln Lys
Pro Asp Gln Ala Pro Lys Leu Leu 595 600 605Ile Lys Tyr Ala Ser Gln
Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser 610 615 620Gly Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu625 630 635 640Ala
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asp Gly His Ser Trp Pro 645 650
655Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser
660 665 670Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly
Gln Val 675 680 685Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala Ser Val 690 695 700Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Ser Ser Tyr Trp Met705 710 715 720His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Ile Gly Glu 725 730 735Ile Asn Pro Gly Asn
Gly His Thr Asn Tyr Asn Glu Lys Phe Lys Ser 740 745 750Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu 755 760 765Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 770 775
780Ser Phe Lys Thr Ala Arg Ala Phe Ala Tyr Trp Gly Gln Gly Thr
Leu785 790 795 800Val Thr Val Ser Ser 80516226PRTArtificial
SequenceFc 16Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Ala
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135
140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro
Gly22517119PRTArtificial Sequence94kvt VH 17Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30Trp Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile
Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Ser
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ser Phe Lys Thr Ala Arg Ala Phe Ala Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11518107PRTArtificial
Sequence94kvt VL 18Asp Ile Val Met Thr Gln Ser Pro Ala Phe Leu Ser
Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln
Thr Ile Ser Asp Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Asp Gln
Ala Pro Lys Leu Leu Ile 35 40 45Lys Tyr
Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala65 70 75
80Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asp Gly His Ser Trp Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
1051910PRTArtificial Sequence(G4S)2 19Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser1 5 102015PRTArtificial Sequence(G4S)3 20Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
152118PRTArtificial Sequence218 21Gly Ser Thr Ser Gly Ser Gly Lys
Pro Gly Ser Gly Glu Gly Ser Thr1 5 10 15Lys Gly22244PRTArtificial
Sequence94kvt HLC 218 22Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Ser Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro Gly Asn Gly
His Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Ser Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Phe
Lys Thr Ala Arg Ala Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro 115 120
125Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Val Met Thr Gln Ser
130 135 140Pro Ala Phe Leu Ser Val Thr Pro Gly Glu Lys Val Thr Ile
Thr Cys145 150 155 160Arg Ala Ser Gln Thr Ile Ser Asp Tyr Leu His
Trp Tyr Gln Gln Lys 165 170 175Pro Asp Gln Ala Pro Lys Leu Leu Ile
Lys Tyr Ala Ser Gln Ser Ile 180 185 190Ser Gly Ile Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Phe Thr Ile Ser
Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr 210 215 220Cys Gln Asp
Gly His Ser Trp Pro Pro Thr Phe Gly Gln Gly Thr Lys225 230 235
240Leu Glu Ile Lys23244PRTArtificial Sequence94kvt LHC 218 23Asp
Ile Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10
15Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr
20 25 30Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Leu Leu
Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
Leu Glu Ala65 70 75 80Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asp Gly
His Ser Trp Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys Gly Ser Thr Ser Gly 100 105 110Ser Gly Lys Pro Gly Ser Gly Glu
Gly Ser Thr Lys Gly Gln Val Gln 115 120 125Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala Ser Val Lys 130 135 140Leu Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met His145 150 155 160Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile 165 170
175Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe Lys Ser Arg
180 185 190Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr Met
Glu Leu 195 200 205Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys Ala Arg Ser 210 215 220Phe Lys Thr Ala Arg Ala Phe Ala Tyr Trp
Gly Gln Gly Thr Leu Val225 230 235 240Thr Val Ser Ser
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